Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Jun 19;4(3):e00350-19.
doi: 10.1128/mSphere.00350-19.

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

Isabella Luísa da Silva Gurgel  1 , Karina Talita de Oliveira Santana Jorge  1 , Nathália Luísa Sousa de Oliveira Malacco  1 , Jéssica Amanda Marques Souza  1 , Marina Campos Rocha  2 , Marina Faria Fernandes  1 , Flávia Rayssa Braga Martins  1 , Iran Malavazi  2 , Mauro Martins Teixeira  3 , Frederico Marianetti Soriani  4
Affiliations

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

Isabella Luísa da Silva Gurgel et al. mSphere. .

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.IMPORTANCEAspergillus 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.

Keywords: Aspergillus fumigatus; cell wall integrity; immune response; msb2; mucin; virulence.

PubMed Disclaimer

Figures

FIG 1
FIG 1
Domain architecture prediction and knockout strain construction. (A) A. fumigatus MsbA shares common features of signaling mucins with A. nidulans MSBA, S. cerevisiae MSB2, C. albicans MSB2, and F. oxysporum MSB2. All of these mucin proteins contain a cleaved signal peptide (SignalP) and one transmembrane domain (TMHMM) close to the C terminus. The large extracellular part is Ser/Thr rich (ProtParam; Color Protein Sequence). Right after the transmembrane region, the short cytoplasmic tail contains a positively charged motif (RR-RKKR--HRR in A. fumigatus; RR-RKKR--HRR in A. nidulans; RRR in S. cerevisiae; RK-RK in C. albicans; and RR-KRKK---HRR in F. oxysporum). (B) The msbA gene was replaced, through homologous recombination, by the auxotrophic marker pyrG. (C) Southern blotting of wild-type strain presenting a fragment of 1.34 kb and the knockout strain (ΔmsbA) presenting a fragment of 3.45 kb.
FIG 2
FIG 2
The ΔmsbA strain has reduced vegetative growth and conidiation. The mutant strain has a diminished radial growth at 30°C, 37°C, and 42°C compared to wild type, in both MM and YAG. (A) MM for 48 h; (B) YAG for 48 h; (C to E) MM at 30°C, 37°C, and 42°C, respectively; (F to H) YAG at 30°C, 37°C, and 42°C, respectively. Quantification was performed by colony diameter measurement every 24 h during 96 h (C to H). Asterisk represents statistical difference (P < 0.05) from wild type under the same growth condition. (I) Percentage of polarized growth after HU release. Samples of germlings were analyzed at 0 h, 0.5 h, 1 h, 1.5 h, and 2 h after release of HU blockade. Asterisk represents statistical difference (P < 0.05) from wild type under the same growth condition. (J) Number of conidia/colony diameter after growth in MM at 37°C for 72 h. Asterisk represents statistical difference (P < 0.05) from wild type.
FIG 3
FIG 3
The ΔmsbA strain shows enhanced adhesion properties and biofilm formation. (A) Optical microscopy of conidiophores from wild-type and ΔmsbA strains. On the right side are shown two independent experiments on wild-type conidiophores. On the left side are shown two independent experiments on ΔmsbA conidiophores. The coverslips were stained using lactophenol blue solution and analyzed by optical microscopy using ×40 magnification. Bars, 50 μm. Experiments were performed in triplicate. (B) Adhesion properties of conidia were analyzed after 4 h of growth in RPMI at 37°C. Adhered conidium number was determined in at least 6 microscope fields and expressed as total percent conidia in each assay. (C) Biofilm formation was measured indirectly by absorbance at 570 nm. Absorbance values were normalized by growth rate of each strain in MM. Values are shown as mean ± SEM. Asterisks represent statistical difference (P < 0.05) from wild type under the same growth condition.
FIG 4
FIG 4
The ΔmsbA strain has altered sensitivity to cell wall stressors and antifungal agents. (A) Tenfold dilution dropout growth in solid MM plates supplemented with different cell wall-perturbing agents: Congo red (CR), calcofluor white (CFW), and NaCl. (B) Conidia (1 × 104 of each strain) were incubated in liquid MM at 37°C for 48 h in the presence of the indicated concentrations of nikkomycin Z. (C) Antifungal susceptibility using Etest gradient strips for voriconazole, itraconazole, and caspofungin. (D) MICs of antifungal drugs analyzed in panel C.
FIG 5
FIG 5
The ΔmsbA strain has altered cell wall thickness. (A) Transmission electronic microscopy images of hyphae from wild-type and ΔmsbA strains grown in liquid MM with or without CFW (100 μg/ml). Bars, 500 nm. (B) Cell wall thickness was quantified under each condition. Values are shown as mean ± SEM from 50 different sections. Asterisk represents statistical difference (P < 0.05) compared with wild type under the same growth condition. Number sign represents statistical difference (P < 0.05) compared within the group (wild type × wild type/ΔmsbA strain × ΔmsbA strain) under control condition.
FIG 6
FIG 6
The ΔmsbA strain has decreased levels of expression of cell wall integrity pathway genes. Strains were grown in YG medium for 24 h at 37°C and treated with CFW (100 μg/ml) for 10 and 30 min. The control group was not treated with CFW. Fold increase in each strain represents the normalized mRNA abundance relative to the wild-type strain under the control condition. Values are shown as mean ± SEM. Asterisk represents statistical difference (P < 0.05) from wild type under the same growth condition. Number sign represents statistical difference (P < 0.05) compared within the group (wild type × wild type/ΔmsbA strain × ΔmsbA strain) under the control condition.
FIG 7
FIG 7
MsbA contributes to downmodulating the inflammatory response after A. fumigatus infection. (A) msbA mRNA expression during infection. A. fumigatus cells were harvested from lungs of infected animals after 4 h and 12 h (PI, postinfection). mRNA was extracted for qRT-PCR assay. Data are presented as mean ± SEM (n = 3 to 4 mice per group). *, significantly different (P < 0.05). (B) Lethality of mice infected with wild-type or ΔmsbA strain. Mice were infected intranasally with 40 μl of suspension containing 1 × 108 conidia of wild-type or ΔmsbA strain, and mortality was monitored for 7 days. NI, noninfected. (C to G) Inflammatory infiltrate was analyzed in BALF of infected animals. Infection with ΔmsbA strain altered inflammatory cells recruitment into airways. Intranasally infected mice had BALFs harvested at day 1 postinfection for inflammatory cell infiltrate determination. Total cell (C), macrophage (D), neutrophil (E), lymphocyte (F), and eosinophil (G) absolute counts in BALF. Data are presented as mean ± SEM (n = 5 to 8 mice per group). *, significantly different (P < 0.05) compared to wild-type-infected group. #, significantly different (P < 0.05) compared to noninfected group. (H) In vitro phagocytosis was performed with RAW 264.7 immortalized macrophages challenged with wild-type or ΔmsbA strain for 2 h and 4 h for phagocytosis. The relationship between the number of macrophages containing conidia inside and the total number of macrophages was used to calculate phagocytic index. One hundred macrophages were counted for each coverslip. (I) Fungal survival was quantified as CFU/phagocytic index (PI). ΔmsbA conidium number after 6 h of phagocytosis was higher. *, significantly different (P < 0.05) compared to wild type.
FIG 8
FIG 8
The ΔmsbA strain stimulates fewer inflammatory mediators in a model of A. fumigatus acute lung infection. Mice were infected with wild-type or ΔmsbA strains, and inflammatory mediators were analyzed by ELISA in BALFs at day 1 postinfection: TNF-α (A), IL-1β (B), IL-10 (C), and CCL11/eotaxin (D). *, significantly different (P < 0.05) from wild-type-infected group. #, significantly different (P < 0.05) from noninfected (NI) group.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Latgé J-P. 2010. Tasting the fungal cell wall. Cell Microbiol 12:863–872. doi:10.1111/j.1462-5822.2010.01474.x. - DOI - PubMed
    1. Rocha MC, Godoy KF, de Castro PA, Hori JI, Bom VLP, Brown NA, Cunha AF, Goldman GH, Malavazi I. 2015. The Aspergillus fumigatus pkcA G579R mutant is defective in the activation of the cell wall integrity pathway but is dispensable for virulence in a neutropenic mouse infection model. PLoS One 10:e0135195. doi:10.1371/journal.pone.0135195. - DOI - PMC - PubMed
    1. Rocha MC, Fabri J, de Godoy KF, de Castro PA, Hori JI, da Cunha AF, Arentshorst M, Ram AFJ, van den Hondel C, Goldman GH, Malavazi I. 2016. Aspergillus fumigatus MADS-box transcription factor rlmA is required for regulation of the cell wall integrity and virulence. G3 (Bethesda) 6:2983–3002. doi:10.1534/g3.116.031112. - DOI - PMC - PubMed
    1. Cullen PJ, Sabbagh W Jr, Graham E, Irick MM, van Olden EK, Neal C, Delrow J, Bardwell L, Sprague GF Jr.. 2004. A signaling mucin at the head of the Cdc42- and MAPK-dependent filamentous growth pathway in yeast. Genes Dev 18:1695–1708. doi:10.1101/gad.1178604. - DOI - PMC - PubMed
    1. Cullen PJ. 2007. Signaling mucins: the new kids on the MAPK block. Crit Rev Eukaryot Gene Expr 17:241–257. doi:10.1615/CritRevEukarGeneExpr.v17.i3.50. - DOI - PubMed

Publication types

MeSH terms

LinkOut - more resources