The Cell Wall Integrity Pathway Contributes to the Early Stages of Aspergillus fumigatus Asexual Development

Abstract

Aspergillus fumigatus is a major cause of human disease. The survival of this fungus is dependent on the cell wall organization and function of its components. The cell wall integrity pathway (CWIP) is the primary signaling cascade that controls de novo synthesis of the cell wall in fungi. Abundant conidiation is a hallmark in A. fumigatus, and uptake of conidia by a susceptible host is usually the initial event in infection. The formation of conidia is mediated by the development of fungus-specific specialized structures, conidiophores, which are accompanied by cell wall remodeling. The molecular regulation of these changes in cell wall composition required for the rise of conidiophore from the solid surface and to disperse the conidia into the air is currently unknown. Here, we investigated the role of CWIP in conidiation. We show that CWIP pkcA^G579R, ΔmpkA, and ΔrlmA mutants displayed reduced conidiation during synchronized asexual differentiation. The transcription factor RlmA directly regulated the expression of regulators of conidiation, including flbB, flbC, brlA, abaA, and rasB, as well as genes involved in cell wall synthesis and remodeling, and this affected the chitin content in aerial hyphae. Phosphorylation of RlmA and MpkA was increased during asexual differentiation. We also observed that MpkA physically associated with the proteins FlbB, FlbC, BrlA, and RasB during this process, suggesting another level of cross talk between the CWIP and asexual development pathways. In summary, our results support the conclusion that one function of the CWIP is the regulation of asexual development in filamentous fungi.IMPORTANCE A remarkable feature of the human pathogen Aspergillus fumigatus is its ability to produce impressive amounts of infectious propagules known as conidia. These particles reach immunocompromised patients and may initiate a life-threatening mycosis. The conidiation process in Aspergillus is governed by a sequence of proteins that coordinate the development of conidiophores. This process requires the remodeling of the cell wall so that the conidiophores can rise and withstand the chains of conidia. The events regulating cell wall remodeling during conidiation are currently unknown. Here, we show that the cell wall integrity pathway (CWIP) components RlmA and MpkA directly contribute to the activation of the conidiation cascade by enabling transcription or phosphorylation of critical proteins involved in asexual development. This study points to an essential role for the CWIP during conidiation and provides further insights into the complex regulation of asexual development in filamentous fungi.

Keywords: Aspergillus fumigatus; MpkA; PkcA; RlmA; asexual development; cell wall integrity.

FIG 1

The CWIP mutants have delayed conidiation when asexual differentiation is induced and altered hydrophobicity of conidia. Conidia were counted by sampling the mycelium of each strain subjected to synchronized asexual differentiation at 2-h intervals at 30°C (A) and 37°C (B). The results are the averages ± the standard deviations (SD; n = 3; *, P ≤ 0.01 for all time points). Absolute values of quantification were log₂ transformed and raw data are available in Data Set S1. (C) Altered hydrophobic properties of conidia in a 1:1 water-oil (tributyrin) interface. (D) Silver-stained SDS-PAGE showing the hydrophobin content extracted from resting conidia. RodA corresponds to the native RodA, and RodA* corresponds to partially degraded or processed RodA (according to reference 50).

FIG 2

CWIP is activated during asexual development and is accompanied by RlmA phosphorylation. (A and B) Western blotting assay of MpkA phosphorylation in a time course experiment of synchronized asexual differentiation. α-(P)-p44/42 or α-p44/42 antibodies were used to detect the phosphorylation and total MpkA, respectively. α-γ-Tubulin antibody was used as a loading control. Signal intensities were quantified, and the ratios of (P)-MpkA to MpkA were calculated and are shown below the panels. (C) Western blot of protein from the rlmA::3×FLAG treated (+ CIP) or not (− CIP) with calf intestinal alkaline phosphatase and probed with α-FLAG antibody in membranes obtained from Phos-tag or regular 8% SDS-PAGE. The arrows point to the phosphorylated forms of RlmA, and the arrowhead points to the unphosphorylated protein. Predicted protein sizes on blot: MpkA, 48.5 kDa; and RlmA::3×FLAG, 70.3 kDa.

FIG 3

The CWIP mutant strains present lower mRNA abundance of genes encoding the central regulators of asexual development. Expression of brlA, abaA, and wetA was investigated by RT-qPCR in the strains subjected to synchronized asexual differentiation during the indicated time points (hours) at 30°C (A) and 37°C (B). Values represent the averages of the results from three independent experiments with two technical repetitions each (see Fig. S6; *P ≤ 0.01 one-way ANOVA), presented as the relative expression of the mutant strain compared to the same time point of the wild-type strain.

FIG 4

RlmA binds to the promoter region of central regulators of conidiation. (A) Location of RlmA binding motifs at the promoter region of the tested genes. Green letters indicate the DNA core sequence 5′-TAWWWWTA-3′ (W = A or T), while black letters indicate the described variations “Y” and “R” at the 5′ and 3′ (1st and 10th base) regions of the motif (Y = C or T; R = A or G). Blue letters indicate a single base variation observed in the binding motif at the promoter region of the brlA and abaA genes. (B) ChIP-qPCR assays of the wild-type and rlmA::GFP strains subjected to synchronized asexual differentiation. Graphs show the average ± the SD from three independent biological experiments (with two technical repetitions each). The percent input is the ratio of the signals obtained from the immunoprecipitated sample and the starting DNA material (input sample) used for the ChIP reaction. Statistical analysis was performed using a one-tailed, paired t test compared to the wild-type control condition (*, P ≤ 0.0001; #, P ≤ 0.005). As a negative binding control, an oligonucleotide located at an exonic region of the prxB gene was used (47).

FIG 5

CWIP genes are required for the expression of flbB and flbC. The expression of flbB, flbC, and rasB was investigated by RT-qPCR in the strains subjected to synchronized asexual differentiation during the indicated time points (hours) at 30°C (A) and 37°C (B). Values represent the averages of results from three independent experiments with two technical repetitions each (see Fig. S7; *, P ≤ 0.01 one-way ANOVA) and are presented as the relative expression of the mutant strain compared to the same time point of the wild-type strain.

FIG 6

MpkA interacts with FlbB, FlbC, BrlA, and RasB during the early stages of conidiation. Co-IP was performed with total protein extracts from the wild-type and the respective strains expressing both GFP and the 3×HA C-terminal tag subjected to synchronized asexual differentiation at 37°C. (A and B) Co-IP experiments performed with an α-GFP antibody that specifically isolates the MpkA 3×HA protein only in the presence of the GFP-tagged proteins. (C and D) Reciprocal Co-IP experiments using an α-HA antibody to isolate the corresponding GFP-tagged proteins. α-Pgk1 was used as an input control. Predicted fusion protein sizes on the blot: FlbB::GFP, 72 kDa; FlbC::GFP, 63.2 kDa; BrlA::GFP, 74.9 kDa; RasB::GFP, 51.5 kDa; and MpkA::3×HA, 51 kDa.

FIG 7

RlmA binds to the promoter region of chitinase and glucanase genes during conidiation. (A) Location of predicted RlmA binding motifs in the promoter region of the tested genes. Green letters indicate the DNA core sequence 5′-TAWWWWTA-3′ (W = A or T), and red letters indicate a variation within this core sequence. Black letters indicate the described variation at Y and R position (1st and 10th base) regions of the motif (Y = C or T; R = A or G). Blue letters indicate a variation observed at the Y or R position. (B) ChIP-qPCR assays of the wild-type and rlmA::GFP strains subjected to synchronized asexual differentiation. Graphs show the averages ± the SD from three independent biological experiments (with two technical repetitions each). Statistical analysis was performed using a one-tailed, paired t test compared to the control condition (*, P ≤ 0.0001; #, P ≤ 0.005).

FIG 8

The CWI pathway contributes to the regulation of the conidiation onset and cell wall remodeling during asexual differentiation. The CWI pathway is activated during the initial stages of asexual competence and culminates with the phosphorylation and activation of the MAP kinase MpkA (red circles). MpkA migrates to the nucleus and phosphorylates RlmA (the details on the interaction between MpkA and RlmA will be published elsewhere). Active RlmA participates in the control of the metabolic changes that take place during the asexual differentiation via the activation of two distinct sets of genes. (i) RlmA binds to the promoter regions of genes involved in conidiation, including the UDAs flbB and flbC and the CRP genes brlA and abaA, allowing timely transcription of such regulators. In addition, the Ras signaling pathway is activated late in the cascade. (ii) RlmA binds to the promoter regions of several genes involved in glucan and chitin metabolism. These enzymes can act on the cell wall (not shown) to cause cell wall remodeling aiming to adapt cells to the formation of aerial structures and conidiation or to induce autolysis, thus contributing to the supply of nutrients required for conidiation. In an additional regulatory event, the active MpkA in the cytosol physically associates with proteins involved in the conidiation pathway. Collectively, the combined actions of activated MpkA and RlmA coordinate alterations in the cell wall composition and timely expression of UDAs and CRP, contributing to the formation of conidia and vegetative growth. The organization of the conidiation pathway was based on references (20, 27, 41). This image was created using BioRender.