Aspergillus fumigatus Hsp90 interacts with the main components of the cell wall integrity pathway and cooperates in heat shock and cell wall stress adaptation

Abstract

The initiation of Aspergillus fumigatus infection occurs via dormant conidia deposition into the airways. Therefore, conidial germination and subsequent hyphal extension and growth occur in a sustained heat shock (HS) environment promoted by the host. The cell wall integrity pathway (CWIP) and the essential eukaryotic chaperone Hsp90 are critical for fungi to survive HS. Although A. fumigatus is a thermophilic fungus, the mechanisms underpinning the HS response are not thoroughly described and important to define its role in pathogenesis, virulence and antifungal drug responses. Here, we investigate the contribution of the CWIP in A. fumigatus thermotolerance. We observed that the CWIP components PkcA, MpkA and RlmA are Hsp90 clients and that a PkcA^G579R mutation abolishes this interaction. PkcA^G579R also abolishes MpkA activation in the short-term response to HS. Biochemical and biophysical analyses indicated that Hsp90 is a dimeric functional ATPase, which has a higher affinity for ADP than ATP and prevents MpkA aggregation in vitro. Our data suggest that the CWIP is constitutively required for A. fumigatus to cope with the temperature increase found in the mammalian lung environment, emphasising the importance of this pathway in supporting thermotolerance and cell wall integrity.

Keywords: Aspergillus fumigatus; Hsp90; MpkA; PkcA; cell wall integrity; heat shock.

Figure 1. CWIP genes contribute to thermotolerant growth.

(A) 1×10² conidia of each strain were exposed to temperatures of 45 °C and 50 °C for 12 and 24 h in Petri dishes containing solid MM. Subsequently, the plates were incubated at 37 °C for recovery for 24 h. The percentage of viable colonies was obtained in comparison to the non-HS control (30 °C). (B) The XTT assay was used to measure the metabolic activity of mature biofilm of CWIP mutants. The biofilms were obtained by growing each strain for 22 h in MM at 37 °C in 24-well plates. Biofilms were then incubated for 2 h at 30 °C. Subsequently, the plates were heat-shocked at 48 °C for the indicated times. The results were expressed as mean ± SD, n = 4. * p ≤ 0.01 One-way ANOVA and Dunnett’s posttest.

Figure 2. The CWIP coordinates cell responses to thermal adaptation and PkcA-dependent MpkA phosphorylation is critical to protect cells from HS at 48 °C.

(A-B) The CWIP mutant strains present lower mRNA abundance of genes encoding the major heat shock proteins. Expression of pkcA, mpkA, rlmA, hsp30, hsp90, hsfA, hsp70, ssc70, and hscA was investigated by RT-qPCR in the strains subjected to heat shock during the indicated time points (h) at 37 °C (A) and 48 °C (B). Values represent the average log_2-fold change relative to the control cDNA (30 °C) for a specific gene compared to the same time point of the wild-type strain (n=3 with 2 technical repetitions each; see Fig. S1 for statistics). The values were submitted to a hierarchical clustering algorithm (Euclidian distance) by using the WebMeV platform. (C) PkcA localizes at the hyphal tips, septum and sites of new growth under physiological conditions, reallocates to the cytosol after HS and eventually returns to the natural accumulation sites after HS recovery. The conidia of each strain were inoculated in MM and incubated at 37 °C for 10 h and room temperature for 1 h. Media was removed and replaced by fresh pre-warmed media at 48 °C to induce HS. Recovery of the fluorescence was followed by the time indicated in the panels. Experiments were independently repeated at least four times and representative germlings of a single experiment are shown. Fluorescence was artificially colored in yellow to enhance visualization. Scale bars = 5 μm. (D) Violin plot of the tip enrichment scores of pkcA::GFP and pkcA^G579R::GFP strains. The mutant protein has a lower degree of enrichment at hyphal tips under physiological conditions (0 min) while it regains baseline tip localization sooner in comparison to the wild-type isoform. The upper table indicate the percent of hyphal tips containing tip localized PkcA at the population level obtained from the average across biological replicates (n = 4 for pkcA::GFP and n = 6 for pkcA^G579R::GFP). Differences in the return of tip localization between the two alleles is seen at 10- and 12-minutes post-HS. For both metrics, statistics were determined by two-way ANOVA with a Sidak’s multiple comparison test. **** p<0.0001, * p<0.05, n.s. not significant. (E) The strains were cultured for 24 h in MM at 30 °C. Subsequently, the mycelium was transferred to fresh pre-heated MM and incubated at 48 °C for the indicated times. The phosphorylated and the total MpkA amount were detected using α-P-p44/42 and α-p44/42 MAPK antibodies, respectively. The α-γ-tubulin antibody was used as loading sample control. Values indicate the ratio of phosphorylated/non-phosphorylated MpkA. Representative images of three independent experiments.

Figure 3. The CWIP mutants are sensitive to Hsp90 inhibition and present late expression of Hsp90 after HS.

(A-B) 1×10³ conidia of the wild-type and CWIP mutants were inoculated in liquid MM supplemented with different concentrations of radicicol and geldanamycin for 48 h at 37 °C. (C-D) Viability after 24 h and 48 h of incubation (37 °C) was quantified by fluorescence of the reduced alamar blue indicator added to each well. Dashed lines indicate the calculated IC₅₀ and shaded areas on each curve indicated the 95% confidence interval. (E-H) Hsp90 protein abundance during HS. The strains were grown for 24 h in MM at 30 °C and subsequently heat-shocked at 48 °C for the indicated timepoints. An α-Hsp90 polyclonal antibody was used to detect Hsp90 and γ-tubulin antibody was used as the loading control. Values indicate the ratio of Hsp90/ γ-tubulin signals. Results are representative images of three independent experiments.

Figure 4.. The CWIP proteins PkcA, MpkA and RlmA are constitutive Hsp90 clients in A. fumigatus.

(A) Domains and structural organization of PkcA sequence. The location of the Gly579, which is mutated to an Ala in the PkcA^G579R mutant, is highlighted in the red box. It is the first residue juxtaposed after the C1B domain in the primary sequence of the polypeptide. The black arrow indicates the C-terminal truncated version of the polypeptide, PkcA(409–1106), expressed in bacterial system (see text for details). PkcA(409–1106) contained the pseudosubstrate site (yellow), the C1 domain, which comprises the cysteine-rich repeats C1A and C1B (green), the fungal specific Q/A/P-rich region (blue) and the catalytic domain of the enzyme (magenta). The structure of PkcA was deduced from Prosite scan and from comparison to other fungi based on (Herrmann et al., 2006, Teepe et al., 2007, Heinisch & Rodicio, 2018). The white segment inside the C-terminal domain indicates the localization of the PxxP region, which is described as necessary for Hsp90 and Cdc37 in mammalian cells (Gould et al., 2009). Identification of PxxP was based in comparison to human PkcA-α (NCBI accession number P17252.4, see text for details). Black and yellow lines indicate the cDNA and polypeptide sequences, respectively. The wild-type and pkcA::3×HA (B), pkcA^G579R::3×HA (C), mpkA::3×HA (D) and rlmA::3×FLAG (E) tagged strains were used in the Co-IP assays. Strains were grown at 30 °C (24 h) and subsequently upshifted to 37 °C or 48 °C for 15 min to induce HS (left panels). To induce CW stress, strains were grown at 37 °C (22 h) and subsequently exposed to 10, 100 or 300 μg/mL of congo red (CR) for 30 min (right panels). Dynabeads protein A bound to the α-HA antibody or α-Flag M2 Affinity gel were used to immunoprecipitated MpkA and PkcA or RlmA, respectively. Co-immunoprecipitated Hsp90 was investigated by Western blot analysis using α-Hsp90 antibody. Hsp90 was also used as the input control for all the samples. Predicted fusion protein sizes on the blot: MpkA::3×HA: 51.4 kDa; PkcA and PkcA^G579R: 127.1 kDa; RlmA::3×FLAG: 70.3; Hsp90: 80.6 kDa.

Figure 5. Protein purification and spectroscopic characterization of Hsp90.

(A) 10% SDS-PAGE showing Hsp90 expression (arrow) in E. coli before and after IPTG induction (lanes 2 and 3, respectively), proteins eluted from Ni⁺²-affinity chromatography (lane 4) and after size exclusion chromatography (SEC; lane 5). The marker of MW is shown in lane 1. (B) CD spectrum of rAfHsp90 acquired in the buffer 40 mM HEPES (pH 7.5), 100 mM KCl, and normalized for mean molar residual ellipticity. (C) Intrinsic fluorescence spectroscopy experiments using the rAfHsp90 protein, in the presence and absence of guanidine hydrochloride (Gnd-HCl). The fluorescence emission spectra reflect the local tertiary environment of the Trp residues. (D) aSEC elution profile for rAfHsp90 and the standard protein mixture. rAfHsp90 eluted with a retention volume similar to apoferritin. Molecular size of standard proteins: apoferritin (480 kDa), γ-globulin (160 kDa), bovine serum albumin (BSA 67 kDa), carbonic anhydrase (30 kDa) and cytochrome C (12.3 kDa). The blue dextran elution profile identifies the void volume. (E) The retention volumes observed for the standard proteins were transformed into the Kav and plotted against the Rs of the standard proteins to determine the Rs for rAfHsp90.

Figure 6. rAfHsp90 binds to the natural ligands ATP and ADP and is a functional ATPase.

The binding of Hsp90 with ATP and ADP was investigated by ITC at 25 °C. ATP (A) or ADP (B) were titrated into the buffer containing the rAfHsp90 generating characteristic thermograms of exothermic reactions. The lower panels indicate the best fitting routine, which yielded values for n, K_A and ΔH_app (C) The calorimetric values for apparent ΔH, ΔG and TΔH resulting from the rAfHsp90 binding to ATP and ADP. The ΔH_app was more than twice higher for ADP binding in comparison to the ATP. (D) Enzyme kinetics of AfHsp90 ATPase activity. AfHsp90 (2 μM) was incubated with ATP (0 – 3 mM) during 90 min at 37 °C and the Pi released from ATP hydrolysis was quantified. The data were treated with a Michaelis-Menten fitting for the achievement of kinetic parameters (graph inset).

Figure 7. rAfHsp90 chaperons rAfMpkA in vitro.

The Hsp90 chaperone activity was assessed by monitoring the ability of rAfHsp90 to prevent aggregation of the client protein by light scattering at 320 nm for 4 h at 42 °C (A) Thermal stability studies of rAfMpkA indicates it aggregates after 120 min of incubation at 42 °C (40 mM HEPES (pH 7.5) buffer containing 100 mM KCl) in a concentration-dependent manner. (B) Inhibition of MpkA aggregation (%) calculated in the presence of 10 μM of rAfMpkA and increasing concentrations of rAfHsp90, i.e: 5 μM (2:1), 7.5 μM (2: 1.3), 10 μM (1:1), 20 μM (1:2), 30 μM (1:3), 40 μM (1:4) and 50 μM (1:5). Aggregation values were calculated considering the maximum aggregation of MpkA as 100%. (D) The human malate dehydrogenase (MDH, 1 μM), a known Hsp90 client, was used as a positive control of rAfHsp90 chaperone activity.

Figure 8. The Hsp90 association with the CWIP components is essential to A. fumigatus thermotolerance.

PkcA is shown in its activated form, according to the S. cerevisiae model. Activation occurs after the dissociation of the interaction between C1, C2 and pseudosubstrate site (ψ), possible after the priming phosphorylation of the protein at the C-terminal kinase (AGC kinase signature) domain (see text for details), by unidentified kinases in A. fumigatus. The domain organization of the protein and color scheme is shown as in Fig. 4A. The HS and congo red (CR) response is thought to emerge mainly from the MidA mechanosensor located at the cell membrane and funneled into Rom2, a guanine nucleotide exchange factor and the small Rho GTPAse Rho1, leading to the activation of PkcA. In the likely absence of DAG stimulation of fungal PkcA via C1 domain, Rho1 can be the linking component that tethers C1A and C1B PkcA domains to the cell membrane, thus suggesting the importance of C1B domain, where the G579R mutation is located (red line). This polypeptide region is important for the interaction of Hsp90 with PkcA since this interaction is abolished in the PkcA^G579R isoform (right). A. fumigatus Rho1 is also an Hsp90 client (data not shown in this study). For all recorded physical interactions, Hsp90 chaperone is shown as a homodimer (green) in its closed state, i.e., bound to ATP since we recorded here all the structural and thermodynamic signatures of this protein and ATPase activity. PkcA activates unknown downstream targets in A. fumigatus and the MAP kinase cascade (green arrow), which ends in the quick activation of MpkA upon HS. MpkA is a constitutive Hsp90 client. The phosphorylation of MpkA is highly dependent on a fully functional PkcA (left). MpkA migrates to the nucleus where it phosphorylates and activates the TF RlmA. RlmA is also an Hsp90 partner and the MpkA/Hsp90/RlmA complex reside in the nucleus under physiological or HS condition. Transcriptional targets of RlmA involved in the CW integrity and HS response are activated. HS (right) stimulates the migration (disappearance) of wild-type and mutated PkcA from the cytosol’s hyphal tips. The return (recruitment) of PkcA^G579R isoform is faster in comparison to the PkcA isoform. Although this model does not explain if PkcA located at the hyphal tip or other cell compartment under basal conditions is enzymatically active, the faster recruitment of PkcA^G579R to the original sites may suggest an inefficient function of the mutated isoform due to the overall alterations in the polypeptide structure that potentially impacts the C1 domain. This diagram is based on data from this article and the following references (Dichtl et al., 2012, Heinisch & Rodicio, 2018, Rocha et al., 2020, Rocha et al., 2016, Rocha et al., 2015, Samantaray et al., 2013, Schmitz & Heinisch, 2003, Valiante et al., 2015). Created with BioRender.com.