Heterotrimeric G-alpha subunits Gpa11 and Gpa12 define a transduction pathway that control spore size and virulence in Mucor circinelloides

Abstract

Mucor circinelloides is one of the causal agents of mucormycosis, an emerging and high mortality rate fungal infection produced by asexual spores (sporangiospores) of fungi that belong to the order Mucorales. M. circinelloides has served as a model genetic system to understand the virulence mechanism of this infection. Although the G-protein signaling cascade plays crucial roles in virulence in many pathogenic fungi, its roles in Mucorales are yet to be elucidated. Previous study found that sporangiospore size and calcineurin are related to the virulence in Mucor, in which larger spores are more virulent in an animal mucormycosis model and loss of a calcineurin A catalytic subunit CnaA results in larger spore production and virulent phenotype. The M. circinelloides genome is known to harbor twelve gpa (gpa1 to gpa12) encoding G-protein alpha subunits and the transcripts of the gpa11 and gpa12 comprise nearly 72% of all twelve gpa genes transcript in spores. In this study we demonstrated that loss of function of Gpa11 and Gpa12 led to larger spore size associated with reduced activation of the calcineurin pathway. Interestingly, we found lower levels of the cnaA mRNAs in sporangiospores from the Δgpa12 and double Δgpa11/Δgpa12 mutant strains compared to wild-type and the ΔcnaA mutant had significantly lower gpa11 and gpa12 mRNA levels compared to wild-type. However, in contrast to the high virulence showed by the large spores of ΔcnaA, the spores from Δgpa11/Δgpa12 were avirulent and produced lower tissue invasion and cellular damage, suggesting that the gpa11 and gpa12 define a signal pathway with two branches. One of the branches controls spore size through regulation of calcineurin pathway, whereas virulences is controlled by an independent pathway. This virulence-related regulatory pathway could control the expression of genes involved in cellular responses important for virulence, since sporangiospores of Δgpa11/Δgpa12 were less resistant to oxidative stress and phagocytosis by macrophages than the ΔcnaA and wild-type strains. The characterization of this pathway could contribute to decipher the signals and mechanism used by Mucorales to produce mucormycosis.

Fig 1. Deletion of gpa11 and gpa12 in M. circinelloides confirmed by Southern-blot.

A) gpa11; B) gpa12; and C) gpa11/gpa12 deletion. The 5′ and 3′ regions (1.1 kb for each) upstream and downstream from the start and stop codons, respectively, of gpa11 or gpa12 were designed to flank the pyrG selection marker for gene deletion (A and B). For the deletion of gpa11 in Δgpa12, the 5′ and 3′ regions (1.1 kb for each) upstream and downstream from the start and stop codons, respectively, of gpa11 were designed to flank leuA (C). For each diagram, the recombinant fragments that were used to transform protoplasts of the strain MU402 (pyrG⁻, leuA⁻) are shown. Molecular confirmation by Southern blotting was performed using specific probes for each gpa gene. DNA samples from transformed and parental (MU402) strains were digested with the indicated restriction enzymes (H, HincII; N, NcoI; P, PvuII).

Fig 2. mRNA quantitation of gpa genes in sporangiospores in gpa11 and gpa12 deletion mutants of M. circinelloides.

Total RNA was isolated from spores of the wild-type, Δgpa11, Δgpa12, and Δgpa11/Δgpa12. The mRNA from each gpa gene was determined by qRT-PCR. Asterisks denote the phylogenetic group of the Gpa proteins in M. circinelloides (* I; **-II, and ***-III) [25]. Four independent experiments were performed under the same conditions. Different letters denote statistically significant differences (ANOVA, Fisher, p<0.05).

Fig 3. Sporangiospore production in Δgpa11 and Δgpa12 mutants of M. circinelloides.

50 spores were grown in YPG agar plates and A) radial growth was recorded each day during the experiment. B) Spores grown after 5 days were counted. Four independent experiments were performed for each condition. Statistically significant differences are indicated by different letters (ANOVA and Fisher’s tests; p ≤ 0.05).

Fig 4. Influence of gpa11 and gpa12 on sporangiospore size in M. circinelloides.

The spores from the different M. circinelloides knockout strains produced in YPG were observed under A) light microscope (100 X), scale bar equal to 20 μm. B) The spore size of each strain was quantified using the Leica Application Suite. C) The spores obtained after five days of incubation on solid YPG media were observed under scanning electron microscope. Representative photographs from the corresponding strains of M. circinelloides under 10,000 magnifications. Scale bar is equal to 1 μm. Statistically significant differences are indicated by different letters (ANOVA and Fisher’s tests; p ≤ 0.05).

Fig 5. Sporangiospore germination of M. circinelloides gpa11 and gpa12 deletion mutants.

A) Percentage of germinated spores in aerobic conditions from each strain in YPG. Aerobic germinated cells were considered independent of hyphae length or number of mother cells. B) Relative expression of pkaR1, quantified by qRT-PCR using tfc-1 as reference gene from hyphae obtained from each strain grown at 2 and 4 h. ΔCT analysis was performed to compare the mRNA levels between strains. Spores from the different strains from M. circinelloides were inoculated into YPG media and incubated with constant shaking (150 rpm) for 3 h, and C) intracellular cAMP levels, and D) PKA activity were determined. E) Percentage of spores germinated under anaerobic conditions in YPG media. F) Relative expression of adh1 determined from yeast cells at 4 h of growth, ΔCT analysis was performed to compare mRNA levels between strains. Four independent experiments were performed for each condition. Statistically significant differences are indicated by different letters (ANOVA and Fisher’s tests; p ≤ 0.05).

Fig 6. Quantitation of cnaA mRNA levels in M. circinelloides Δgpa11 and Δgpa12 mutant strains.

Expression of cnaA relative to tfc-1 in total RNA purified from sporangiospores and mycelium grown in aerobic conditions after indicated times in YPG. qRT-PCR was performed to determine the transcript levels of cnaA and a ΔCt analysis was performed to compare the mRNA level between the strains. Four independent experiments were performed for each condition. Statistically significant differences are indicated by different letters (ANOVA and Fisher’s tests; p ≤ 0.05).

Fig 7. Quantitation of gpa mRNA levels in M. circinelloides ΔcnaA.

Changes in levels of mRNA from different gpa genes from total RNA purified from sporangiospores of wild-type (closed bars) and ΔcnaA (open bars). qRT-PCR was carried out to determine the transcript levels of gpa genes, ΔCt analysis was performed to compare the mRNA levels between the strains. Four independent experiments were performed for each condition. Statistically significant differences are indicated by different letters (ANOVA and Fisher’s tests; p ≤ 0.05).

Fig 8. Influence of Gpa11, Gpa12, and CnaA on mRNA levels of ena genes in M. circinelloides.

Levels of A) ena1 and B) ena2 measured by qRT-PCR from total mRNA from sporangiospores. ΔΔCt analysis was performed to compare mRNA levels between strains. Four independent experiments were performed for each condition. Different letters denote statistically significant differences (ANOVA, Fisher, p<0.05).

Fig 9. Role of gpa11 and gpa12 in the virulence of M. circinelloides.

A) Survival of diabetic mice infected intraperitoneally with 2 × 10⁷ sporangiospores from different M. circinelloides strains recorded each day post-injection. N = 8, two independent experiments. Relative expression of inflammation markers B) IL-6, C) IL-1β, D) MIP-2, E) TNF-α used to monitor tissue damage in the liver and lung of mice infected with spores from the different M. circinelloides strains (N = 4). F) Fungal burden, determined by qPCR, from the liver and lung of infected mice. Three independent experiments were performed for each condition. Statistically significant differences are indicated by different letters (ANOVA and Fisher’s tests; p ≤ 0.05).

Fig 10. Effect of mutations in gpa11 and gpa12 in the viability of sporangiospores from M. circinelloides in H₂O₂ and macrophage interaction.

A) Spores from different strains were treated or not with 4 mM H₂O₂ and incubated at 28 °C on YPG. Survival was obtained from colonies grown for 24 h from the quotient of colonies from treatment versus no treatment. B) Germination of spores during macrophage interaction after 3 h was observed under direct observation by light microscopy (40 X) Scale bar = 20 μm. Arrows indicate germinating hyphae and head of arrows indicate swelling spores. C) The pkaR1 mRNA levels were quantified by qRT-PCR after 1 and 3 h of spore and macrophage interaction. D) Quantitation of killed spores by qPCR using tfc-1 after 3 h of incubation with mouse macrophages. A ΔCt analysis was performed to compare the mRNA and gene levels between the samples. Figures show the average of three independent experiments. Statistically significant differences are indicated by different letters (ANOVA and Fisher’s tests; p ≤ 0.05).

Fig 11. Proposed model of the function of Gpa11 and Gpa12 regulating sporangiospore size and virulence in M. circinelloides.

Through one or more GPCRs, Gpa11 and Gpa12 in the spores of M. circinelloides activate CnaA, which in turn activates the calcineurin pathway. The calcineurin pathway leads to the expression of genes regulated by this pathway, like ena1 and ena2. The dysfunction of Ena1 and Ena2, which are Na⁺-K⁺ ATPases, could increase cytosolic levels of sodium ions, allowing the influx of water, leading to an increase in spore size in M. circinelloides as was observed in the Δgpa11/Δgpa12 strain. The CnaA negatively regulates the virulence and stimulates the PKA pathway, meanwhile Gpa11 or Gpa12 positively regulate the virulence and activate the PKA pathway during aerobic germination, but unknown elements are implied in the avirulent phenotype by the mutations in gpa11 and gpa12.