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 Mitogen-Activated Protein Kinase Cross-Talk Interaction Modulates the Production of Melanins in Aspergillus fumigatus

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Adriana Oliveira Manfiolli ◽  
Filipe Silva Siqueira ◽  
Thaila Fernanda dos Reis ◽  
Patrick Van Dijck ◽  
Sanne Schrevens ◽  
...  
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Cross Talk ◽  
Pathogenic Fungus ◽  
Antifungal Drugs ◽  
Mitogen Activated Protein Kinase ◽  
Melanin Production ◽  
Content Type ◽  
Mitogen Activated Protein ◽  
Salvage Pathways

ABSTRACT The pathogenic fungus Aspergillus fumigatus is able to adapt to extremely variable environmental conditions. The A. fumigatus genome contains four genes coding for mitogen-activated protein kinases (MAPKs), which are important regulatory knots involved in diverse cellular responses. From a clinical perspective, MAPK activity has been connected to salvage pathways, which can determine the failure of effective treatment of invasive mycoses using antifungal drugs. Here, we report the characterization of the Saccharomyces cerevisiae Fus3 ortholog in A. fumigatus, designated MpkB. We demonstrate that MpkB is important for conidiation and that its deletion induces a copious increase of dihydroxynaphthalene (DHN)-melanin production. Simultaneous deletion of mpkB and mpkA, the latter related to maintenance of the cell wall integrity, normalized DHN-melanin production. Localization studies revealed that MpkB translocates into the nuclei when A. fumigatus germlings are exposed to caspofungin stress, and this is dependent on the cross-talk interaction with MpkA. Additionally, DHN-melanin formation was also increased after deletion of genes coding for the Gα protein GpaA and for the G protein-coupled receptor GprM. Yeast two-hybrid and coimmunoprecipitation assays confirmed that GpaA and GprM interact, suggesting their role in the MpkB signaling cascade. IMPORTANCE Aspergillus fumigatus is the most important airborne human pathogenic fungus, causing thousands of deaths per year. Its lethality is due to late and often inaccurate diagnosis and the lack of efficient therapeutics. The failure of efficient prophylaxis and therapy is based on the ability of this pathogen to activate numerous salvage pathways that are capable of overcoming the different drug-derived stresses. A major role in the protection of A. fumigatus is played by melanins. Melanins are cell wall-associated macromolecules classified as virulence determinants. The understanding of the various signaling pathways acting in this organism can be used to elucidate the mechanism beyond melanin production and help to identify ideal drug targets.

scholarly journals PKC1 Is Essential for Protection against both Oxidative and Nitrosative Stresses, Cell Integrity, and Normal Manifestation of Virulence Factors in the Pathogenic Fungus Cryptococcus neoformans

2008 ◽  
Vol 7 (10) ◽  
pp. 1685-1698 ◽  
Author(s):  
Kimberly J. Gerik ◽  
Sujit R. Bhimireddy ◽  
Jan S. Ryerse ◽  
Charles A. Specht ◽  
Jennifer K. Lodge
Keyword(s):  
Cell Wall ◽  
Nitrosative Stress ◽  
Pathogenic Fungus ◽  
Fungal Growth ◽  
Mitogen Activated Protein Kinase ◽  
Cell Integrity ◽  
Wild Type ◽  
Pathway Activation ◽  
Mitogen Activated Protein ◽  
Kinase Cascade

ABSTRACT Cell wall integrity is crucial for fungal growth, survival, and pathogenesis. Responses to environmental stresses are mediated by the highly conserved Pkc1 protein and its downstream components. In this study, we demonstrate that both oxidative and nitrosative stresses activate the PKC1 cell integrity pathway in wild-type cells, as measured by phosphorylation of Mpk1, the terminal protein in the PKC1 phosphorylation cascade. Furthermore, deletion of PKC1 shows that this gene is essential for defense against both oxidative and nitrosative stresses; however, other genes involved directly in the PKC1 pathway are dispensable for protection against these stresses. This suggests that Pkc1 may have multiple and alternative functions other than activating the mitogen-activated protein kinase cascade from a “top-down” approach. Deletion of PKC1 also causes osmotic instability, temperature sensitivity, severe sensitivity to cell wall-inhibiting agents, and alterations in capsule and melanin. Furthermore, the vital cell wall components chitin and its deacetylated form chitosan appear to be mislocalized in a pkc1Δ strain, although this mutant contains wild-type levels of both of these polymers. These data indicate that loss of Pkc1 has pleiotropic effects because it is central to many functions either dependent on or independent of PKC1 pathway activation. Notably, this is the first time that Pkc1 has been implicated in protection against nitrosative stress in any organism.

scholarly journals The Aspergillus fumigatus Phosphoproteome Reveals Roles of High-Osmolarity Glycerol Mitogen-Activated Protein Kinases in Promoting Cell Wall Damage and Caspofungin Tolerance

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Eliciane Cevolani Mattos ◽  
Lilian Pereira Silva ◽  
Clara Valero ◽  
Patrícia Alves de Castro ◽  
Thaila Fernanda dos Reis ◽  
...  
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Protein Kinases ◽  
Wall Stress ◽  
Mapk Signaling ◽  
Fungal Cell ◽  
Content Type ◽  
Cell Wall Stress ◽  
Cell Wall Damage ◽  
Mitogen Activated Protein

ABSTRACT The filamentous fungus Aspergillus fumigatus can cause a distinct set of clinical disorders in humans. Invasive aspergillosis (IA) is the most common life-threatening fungal disease of immunocompromised humans. The mitogen-activated protein kinase (MAPK) signaling pathways are essential to the adaptation to the human host. Fungal cell survival is highly dependent on the organization, composition, and function of the cell wall. Here, an evaluation of the global A. fumigatus phosphoproteome under cell wall stress caused by the cell wall-damaging agent Congo red (CR) revealed 485 proteins potentially involved in the cell wall damage response. Comparative phosphoproteome analyses with the ΔsakA, ΔmpkC, and ΔsakA ΔmpkC mutant strains from the osmotic stress MAPK cascades identify their additional roles during the cell wall stress response. Our phosphoproteomics allowed the identification of novel kinases and transcription factors (TFs) involved in osmotic stress and in the cell wall integrity (CWI) pathway. Our global phosphoproteome network analysis showed an enrichment for protein kinases, RNA recognition motif domains, and the MAPK signaling pathway. In contrast to the wild-type strain, there is an overall decrease of differentially phosphorylated kinases and phosphatases in ΔsakA, ΔmpkC, and ΔsakA ΔmpkC mutants. We constructed phosphomutants for the phosphorylation sites of several proteins differentially phosphorylated in the wild-type and mutant strains. For all the phosphomutants, there is an increase in the sensitivity to cell wall-damaging agents and a reduction in the MpkA phosphorylation upon CR stress, suggesting these phosphosites could be important for the MpkA modulation and CWI pathway regulation. IMPORTANCE Aspergillus fumigatus is an opportunistic human pathogen causing allergic reactions or systemic infections, such as invasive pulmonary aspergillosis in immunocompromised patients. The mitogen-activated protein kinase (MAPK) signaling pathways are essential for fungal adaptation to the human host. Fungal cell survival, fungicide tolerance, and virulence are highly dependent on the organization, composition, and function of the cell wall. Upon cell wall stress, MAPKs phosphorylate multiple target proteins involved in the remodeling of the cell wall. Here, we investigate the global phosphoproteome of the ΔsakA and ΔmpkC A. fumigatus and high-osmolarity glycerol (HOG) pathway MAPK mutants upon cell wall damage. This showed the involvement of the HOG pathway and identified novel protein kinases and transcription factors, which were confirmed by fungal genetics to be involved in promoting tolerance of cell wall damage. Our results provide understanding of how fungal signal transduction networks modulate the cell wall. This may also lead to the discovery of new fungicide drug targets to impact fungal cell wall function, fungicide tolerance, and virulence.

scholarly journals Aspergillus fumigatus Transcription Factors Involved in the Caspofungin Paradoxical Effect

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Clara Valero ◽  
Ana Cristina Colabardini ◽  
Jéssica Chiaratto ◽  
Lakhansing Pardeshi ◽  
Patrícia Alves de Castro ◽  
...  
Keyword(s):  
Cell Wall ◽  
Transcription Factors ◽  
Invasive Aspergillosis ◽  
Aspergillus Fumigatus ◽  
Rna Polymerase Ii ◽  
Respiratory Function ◽  
Antifungal Agents ◽  
Fungal Species ◽  
Content Type ◽  
Cell Wall Remodeling

ABSTRACT Aspergillus fumigatus is the leading cause of pulmonary fungal diseases. Azoles have been used for many years as the main antifungal agents to treat and prevent invasive aspergillosis. However, in the last 10 years there have been several reports of azole resistance in A. fumigatus and new strategies are needed to combat invasive aspergillosis. Caspofungin is effective against other human-pathogenic fungal species, but it is fungistatic only against A. fumigatus. Resistance to caspofungin in A. fumigatus has been linked to mutations in the fksA gene that encodes the target enzyme of the drug β-1,3-glucan synthase. However, tolerance of high caspofungin concentrations, a phenomenon known as the caspofungin paradoxical effect (CPE), is also important for subsequent adaptation and drug resistance evolution. Here, we identified and characterized the transcription factors involved in the response to CPE by screening an A. fumigatus library of 484 null transcription factors (TFs) in CPE drug concentrations. We identified 11 TFs that had reduced CPE and that encoded proteins involved in the basal modulation of the RNA polymerase II initiation sites, calcium metabolism, and cell wall remodeling. One of these TFs, FhdA, was important for mitochondrial respiratory function and iron metabolism. The ΔfhdA mutant showed decreased growth when exposed to Congo red or to high temperature. Transcriptome sequencing (RNA-seq) analysis and further experimental validation indicated that the ΔfhdA mutant showed diminished respiratory capacity, probably affecting several pathways related to the caspofungin tolerance and resistance. Our results provide the foundation to understand signaling pathways that are important for caspofungin tolerance and resistance. IMPORTANCE Aspergillus fumigatus, one of the most important human-pathogenic fungal species, is able to cause aspergillosis, a heterogeneous group of diseases that presents a wide range of clinical manifestations. Invasive pulmonary aspergillosis is the most serious pathology in terms of patient outcome and treatment, with a high mortality rate ranging from 50% to 95% primarily affecting immunocompromised patients. Azoles have been used for many years as the main antifungal agents to treat and prevent invasive aspergillosis. However, there were several reports of evolution of clinical azole resistance in the last decade. Caspofungin, a noncompetitive β-1,3-glucan synthase inhibitor, has been used against A. fumigatus, but it is fungistatic and is recommended as second-line therapy for invasive aspergillosis. More information about caspofungin tolerance and resistance is necessary in order to refine antifungal strategies that target the fungal cell wall. Here, we screened a transcription factor (TF) deletion library for TFs that can mediate caspofungin tolerance and resistance. We have identified 11 TFs that are important for caspofungin sensitivity and/or for the caspofungin paradoxical effect (CPE). These TFs encode proteins involved in the basal modulation of the RNA polymerase II initiation sites, calcium metabolism or cell wall remodeling, and mitochondrial respiratory function. The study of those genes regulated by TFs identified in this work will provide a better understanding of the signaling pathways that are important for caspofungin tolerance and resistance.

scholarly journals The Aspergillus fumigatus sitA Phosphatase Homologue Is Important for Adhesion, Cell Wall Integrity, Biofilm Formation, and Virulence

2015 ◽  
Vol 14 (8) ◽  
pp. 728-744 ◽  
Author(s):  
Vinícius Leite Pedro Bom ◽  
Patrícia Alves de Castro ◽  
Lizziane K. Winkelströter ◽  
Marçal Marine ◽  
Juliana I. Hori ◽  
...  
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Biofilm Formation ◽  
Essential Gene ◽  
Pathogenic Fungus ◽  
Invasive Pulmonary Aspergillosis ◽  
Cell Wall Integrity ◽  
Necrosis Factor Alpha ◽  
Content Type ◽  
Opportunistic Pathogenic

ABSTRACTAspergillus fumigatusis an opportunistic pathogenic fungus able to infect immunocompromised patients, eventually causing disseminated infections that are difficult to control and lead to high mortality rates. It is important to understand how the signaling pathways that regulate these factors involved in virulence are orchestrated. Protein phosphatases are central to numerous signal transduction pathways. Here, we characterize theA. fumigatusprotein phosphatase 2A SitA, theSaccharomyces cerevisiaeSit4p homologue. ThesitAgene is not an essential gene, and we were able to construct anA. fumigatusnull mutant. The ΔsitAstrain had decreased MpkA phosphorylation levels, was more sensitive to cell wall-damaging agents, had increased β-(1,3)-glucan and chitin, was impaired in biofilm formation, and had decreased protein kinase C activity. The ΔsitAstrain is more sensitive to several metals and ions, such as MnCl2, CaCl2, and LiCl, but it is more resistant to ZnSO4. The ΔsitAstrain was avirulent in a murine model of invasive pulmonary aspergillosis and induces an augmented tumor necrosis factor alpha (TNF-α) response in mouse macrophages. These results stress the importance ofA. fumigatusSitA as a possible modulator of PkcA/MpkA activity and its involvement in the cell wall integrity pathway.

scholarly journals Putative Membrane Receptors Contribute to Activation and Efficient Signaling of Mitogen-Activated Protein Kinase Cascades during Adaptation of Aspergillus fumigatus to Different Stressors and Carbon Sources

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Lilian Pereira Silva ◽  
Dean Frawley ◽  
Leandro José de Assis ◽  
Ciara Tierney ◽  
Alastair B. Fleming ◽  
...  
Keyword(s):  
Cell Wall ◽  
Protein Kinase ◽  
Aspergillus Fumigatus ◽  
Fungal Pathogen ◽  
Cell Wall Integrity ◽  
Mitogen Activated Protein Kinase ◽  
Hog Pathway ◽  
Content Type ◽  
High Osmolarity Glycerol ◽  
Mitogen Activated Protein

ABSTRACT The high-osmolarity glycerol (HOG) response pathway is a multifunctional signal transduction pathway that specifically transmits ambient osmotic signals. Saccharomyces cerevisiae Hog1p has two upstream signaling branches, the sensor histidine kinase Sln1p and the receptor Sho1p. The Sho1p branch includes two other proteins, the Msb2p mucin and Opy2p. Aspergillus fumigatus is the leading cause of pulmonary fungal diseases. Here, we investigated the roles played by A. fumigatus SlnASln1p, ShoASho1p, MsbAMsb2p, and OpyAOpy2p putative homologues during the activation of the mitogen-activated protein kinase (MAPK) HOG pathway. The shoA, msbA, and opyA singly and doubly null mutants are important for the cell wall integrity (CWI) pathway, oxidative stress, and virulence as assessed by a Galleria mellonella model. Genetic interactions of ShoA, MsbA, and OpyA are also important for proper activation of the SakAHog1p and MpkASlt2 cascade and the response to osmotic and cell wall stresses. Comparative label-free quantitative proteomics analysis of the singly null mutants with the wild-type strain upon caspofungin exposure indicates that the absence of ShoA, MsbA, and OpyA affects the osmotic stress response, carbohydrate metabolism, and protein degradation. The putative receptor mutants showed altered trehalose and glycogen accumulation, suggesting a role for ShoA, MsbA, and OpyA in sugar storage. Protein kinase A activity was also decreased in these mutants. We also observed genetic interactions between SlnA, ShoA, MsbA, and OpyA, suggesting that both branches are important for activation of the HOG/CWI pathways. Our results help in the understanding of the activation and modulation of the HOG and CWI pathways in this important fungal pathogen. IMPORTANCE Aspergillus fumigatus is an important human-pathogenic fungal species that is responsible for a high incidence of infections in immunocompromised individuals. A. fumigatus high-osmolarity glycerol (HOG) and cell wall integrity pathways are important for the adaptation to different forms of environmental adversity such as osmotic and oxidative stresses, nutrient limitations, high temperatures, and other chemical and mechanical stresses that may be produced by the host immune system and antifungal drugs. Little is known about how these pathways are activated in this fungal pathogen. Here, we characterize four A. fumigatus putative homologues that are important for the activation of the yeast HOG pathway. A. fumigatus SlnASln1p, ShoASho1p, MsbAMsb2p, and OpyAOpy2p are genetically interacting and are essential for the activation of the HOG and cell wall integrity pathways. Our results contribute to the understanding of A. fumigatus adaptation to the host environment.

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 Proteome Analysis Reveals the Conidial Surface Protein CcpA Essential for Virulence of the Pathogenic FungusAspergillus fumigatus

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Vera Voltersen ◽  
Matthew G. Blango ◽  
Sahra Herrmann ◽  
Franziska Schmidt ◽  
Thorsten Heinekamp ◽  
...  
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Surface Structure ◽  
Fungal Spores ◽  
Fungal Spore ◽  
Invasive Infection ◽  
Immune Recognition ◽  
Wild Type ◽  
Content Type ◽  
Conidial Surface

ABSTRACTAspergillus fumigatusis a common airborne fungal pathogen of humans and a significant source of mortality in immunocompromised individuals. Here, we provide the most extensive cell wall proteome profiling to date ofA. fumigatusresting conidia, the fungal morphotype pertinent to first contact with the host. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified proteins within the conidial cell wall by hydrogen-fluoride (HF)–pyridine extraction and proteins exposed on the surface using a trypsin-shaving approach. One protein, designatedconidialcell wallproteinA(CcpA), was identified by both methods and was found to be nearly as abundant as hydrophobic rodlet layer-forming protein RodA. CcpA, an amphiphilic protein, like RodA, peaks in expression during sporulation on resting conidia. Despite high cell wall abundance, the cell surface structure of ΔccpAresting conidia appeared normal. However, trypsin shaving of ΔccpAconidia revealed novel surface-exposed proteins not detected on conidia of the wild-type strain. Interestingly, the presence of swollen ΔccpAconidia led to higher activation of neutrophils and dendritic cells than was seen with wild-type conidia and caused significantly less damage to epithelial cellsin vitro. In addition, virulence was highly attenuated when cortisone-treated, immunosuppressed mice were infected with ΔccpAconidia. CcpA-specific memory T cell responses were detectable in healthy human donors naturally exposed toA. fumigatusconidia, suggesting a role for CcpA as a structural protein impacting conidial immunogenicity rather than possessing a protein-intrinsic immunosuppressive effect. Together, these data suggest that CcpA serves as a conidial stealth protein by altering the conidial surface structure to minimize innate immune recognition.IMPORTANCEThe mammalian immune system relies on recognition of pathogen surface antigens for targeting and clearance. In the absence of immune evasion strategies, pathogen clearance is rapid. In the case ofAspergillus fumigatus, the successful fungus must avoid phagocytosis in the lung to establish invasive infection. In healthy individuals, fungal spores are cleared by immune cells; however, in immunocompromised patients, clearance mechanisms are impaired. Here, using proteome analyses, we identified CcpA as an important fungal spore protein involved in pathogenesis.A. fumigatuslacking CcpA was more susceptible to immune recognition and prompt eradication and, consequently, exhibited drastically attenuated virulence. In infection studies, CcpA was required for virulence in infected immunocompromised mice, suggesting that it could be used as a possible immunotherapeutic or diagnostic target in the future. In summary, our report adds a protein to the list of those known to be critical to the complex fungal spore surface environment and, more importantly, identifies a protein important for conidial immunogenicity during infection.

scholarly journals Surface Stress Induces a Conserved Cell Wall Stress Response in the Pathogenic Fungus Candida albicans

2012 ◽  
Vol 12 (2) ◽  
pp. 254-264 ◽  
Author(s):  
Clemens J. Heilmann ◽  
Alice G. Sorgo ◽  
Sepehr Mohammadi ◽  
Grazyna J. Sosinska ◽  
Chris G. de Koster ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Cell Separation ◽  
Pathogenic Fungus ◽  
Wall Stress ◽  
Content Type ◽  
Human Fungal Pathogen ◽  
Mitogen Activated Protein ◽  
Similar Phenotype ◽  
Stressed Cells

ABSTRACTThe human fungal pathogenCandida albicanscan grow at temperatures of up to 45°C. Here, we show that at 42°C substantially less biomass was formed than at 37°C. The cells also became more sensitive to wall-perturbing compounds, and the wall chitin levels increased, changes that are indicative of wall stress. Quantitative mass spectrometry of the wall proteome using15N metabolically labeled wall proteins as internal standards revealed that at 42°C the levels of the β-glucan transglycosylases Phr1 and Phr2, the predicted chitin transglycosylases Crh11 and Utr2, and the wall maintenance protein Ecm33 increased. Consistent with our previous results for fluconazole stress, this suggests that a wall-remodeling response is mounted to relieve wall stress. Thermal stress as well as different wall and membrane stressors led to an increased phosphorylation of the mitogen-activated protein (MAP) kinase Mkc1, suggesting activation of the cell wall integrity (CWI) pathway. Furthermore, all wall and membrane stresses tested resulted in diminished cell separation. This was accompanied by decreased secretion of the major chitinase Cht3 and the endoglucanase Eng1 into the medium. Consistent with this,cht3cells showed a similar phenotype. When treated with exogenous chitinase, cell clusters both from stressed cells and mutant strains were dispersed, underlining the importance of Cht3 for cell separation. We propose that surface stresses lead to a conserved cell wall remodeling response that is mainly governed by Mkc1 and is characterized by chitin reinforcement of the wall and the expression of remedial wall remodeling enzymes.

scholarly journals The Aspergillus fumigatus Mucin MsbA Regulates the Cell Wall Integrity Pathway and Controls Recognition of the Fungus by the Immune System

mSphere ◽  
2019 ◽  
Vol 4 (3) ◽  
Author(s):  
Isabella Luísa da Silva Gurgel ◽  
Karina Talita de Oliveira Santana Jorge ◽  
Nathália Luísa Sousa de Oliveira Malacco ◽  
Jéssica Amanda Marques Souza ◽  
Marina Campos Rocha ◽  
...  
Keyword(s):  
Cell Wall ◽  
Immune System ◽  
Aspergillus Fumigatus ◽  
Fungal Growth ◽  
Cell Wall Composition ◽  
Cell Wall Integrity ◽  
Cell Physiology ◽  
Content Type ◽  
Cell Wall Morphogenesis

ABSTRACT Aspergillus fumigatus is a filamentous fungus which causes invasive pulmonary aspergillosis in immunocompromised individuals. In fungi, cell signaling and cell wall plasticity are crucial for maintaining physiologic processes. In this context, Msb2 is an important signaling mucin responsible for activation of a variety of mitogen-activated protein kinase (MAPK)-dependent signaling pathways that regulate cell growth in several organisms, such as the cell wall integrity (CWI) pathway. Here, we aimed to characterize the MSB2 homologue in A. fumigatus. Our results showed that MsbA plays a role in the vegetative and reproductive development of the fungus, in stress adaptation, and in resistance to antifungal drugs by modulating the CWI pathway gene expression. Importantly, cell wall composition is also responsible for activation of diverse receptors of the host immune system, thus leading to a proper immune response. In a model of acute Aspergillus pulmonary infection, results demonstrate that the ΔmsbA mutant strain induced less inflammation with diminished cell influx into the lungs and lower cytokine production, culminating in increased lethality rate. These results characterize for the first time the role of the signaling mucin MsbA in the pathogen A. fumigatus, as a core sensor for cell wall morphogenesis and an important regulator of virulence. IMPORTANCE Aspergillus fumigatus is an opportunistic fungus with great medical importance. During infection, Aspergillus grows, forming hyphae that colonize the lung tissue and invade and spread over the mammal host, resulting in high mortality rates. The knowledge of the mechanisms responsible for regulation of fungal growth and virulence comprises an important point to better understand fungal physiology and host-pathogen interactions. Msb2 is a mucin that acts as a sensor and an upstream regulator of the MAPK pathway responsible for fungal development in Candida albicans and Aspergillus nidulans. Here, we show the role of the signaling mucin MsbA in the pathogen A. fumigatus, as a core sensor for cell wall morphogenesis, fungal growth, and virulence. Moreover, we show that cell wall composition, controlled by MsbA, is detrimental for fungal recognition and clearance by immune cells. Our findings are important for the understanding of how fungal sensors modulate cell physiology.

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