Trehalose 6-phosphate phosphatase is required for cell wall integrity and fungal virulence but not trehalose biosynthesis in the human fungal pathogen Aspergillus fumigatus

Abstract

The trehalose biosynthesis pathway is critical for virulence in human and plant fungal pathogens. In this study, we tested the hypothesis that trehalose 6-phosphate phosphatase (T6PP) is required for Aspergillus fumigatus virulence. A mutant of the A. fumigatus T6PP, OrlA, displayed severe morphological defects related to asexual reproduction when grown on glucose (1%) minimal media. These defects could be rescued by addition of osmotic stabilizers, reduction in incubation temperature or increase in glucose levels (> 4%). Subsequent examination of the mutant with cell wall perturbing agents revealed a link between cell wall biosynthesis and trehalose 6-phosphate (T6P) levels. As expected, high levels of T6P accumulated in the absence of OrlA resulting in depletion of free inorganic phosphate and inhibition of hexokinase activity. Surprisingly, trehalose production persisted in the absence of OrlA. Further analyses revealed that A. fumigatus contains two trehalose phosphorylases that may be responsible for trehalose production in the absence of OrlA. Despite a normal growth rate under in vitro growth conditions, the orlA mutant was virtually avirulent in two distinct murine models of invasive pulmonary aspergillosis. Our results suggest that further study of this pathway will lead to new insights into regulation of fungal cell wall biosynthesis and virulence.

Fig. 1

Generation of strains used in this study. (A) Locus of orlA gene in wild-type strain (CEA10) and (B) locus of orlA after successful introduction of gene replacement construct (C) Southern blot analysis of wild-type, reconstituted strain (Rec-orlA) and ΔorlA strains. The XbaI digested genomic DNA of all strains were separated on a 1% agarose gel, blotted, and hybridized with a 900 bp genomic DNA probe from the orlA upstream sequence. The expected fragment sizes of the orlA locus in the wild-type and orlA mutant were observed and are 10,270 bp and 4,375 bp respectively. The reconstituted strain (Rec-orlA) contains the expected hybridization signals for a single ectopic insertion of the wild-type allele of orlA (top) and maintenance of the disrupted orlA locus (bottom).

Fig. 2

Colony morphology and conidia production in the absence of OrlA. (A) Normal growth rate but abolished asexual conidiation of the orlA mutant is observed on glucose minimal media (GMM) at 37°C. This defect could be recovered on 1.2M sorbitol minimal media (SMM) at 37°C. (B) Conidia production of wild-type, orlA mutant and orlA reconstituted strain on two different media GMM and SMM incubated at 37°C for 4 days prior to harvesting and counting. Data is the mean and standard deviation of three biological replicates. *Total conidia production of the mutant cultured on GMM is significantly less than the wild-type and reconstituted strains (P = 0.020 and 0.003 respectively).

Fig. 3

Hyphal morphology of the orlA mutant is altered. (A) Slide cultures and lactophenol cotton blue staining reveal dysmorphic hyphae and malformed asexual reproductive structures which formed vesicles lacking phialides in the orlA mutant grown on GMM at 37°C. (B) The ability to form normal hyphae, asexual reproductive structures, and conidia is restored in the orlA mutant grown on GMM containing 10% glucose or minimal media containing 1.2 M sorbitol (SMM) at 37°C. (C) Wild-type CEA10 generates normal hyphae and asexual reproductive structures when grown on GMM and SMM at 37°C. All representative pictures were captured under light microscopy at 400× magnification (reference bar = 50 μm).

Fig. 4

Flow cytometry analysis reveals a heterogeneous population of orlA mutant conidia from SMM when cultured at 37°C while wild-type (CEA10) and reconstituted strains (Rec-orlA) display a homogenous population of conidia. FACS analysis via forward and side scatter parameters of equal amounts (10,000 events) of conidia. Differences in conidia size and shape can be observed via light microscopic observation at 400× magnification. A reference bar is 10 μm length.

Fig. 5

The orlA mutant is sensitive to cell wall perturbing agents. Cell wall defects were observed in the orlA null mutant cultured on GMM at 37°C containing various cell wall inhibitors. (A) Congo Red (CR) 1 mg/ml. (B) Calcofluor White (CFW) 25 μg/ml. (C) Nikkomycin Z (NK) 0.1mM. (D) GMM media without inhibitors was used as a control that represents the normal growth of all strains incubated at 37°C for 3 days. Concentrations presented are the optimum concentrations used to determine the growth defect of the orlA mutant (ΔorlA) compared to the wild-type (CEA10) and reconstituted strains (Rec-orlA). The experiment was repeated in biological triplicates with identical results.

Fig. 6

Production of trehalose and trehalose-6-phosphate (T6P). (A) Conidia for trehalose accumulation measurements were cultured at 37°C and 45°C. At 37°C, trehalose amounts found in the mutant were not significantly different from the wild-type and reconstituted strains; however at 45°C the mutant accumulated greater amounts of trehalose compared to the wild-type and reconstituted strains (*P =0.04 and 0.02 respectively. (B) T6P accumulation in conidia and mycelia were measured with LCMS from cell free culture extracts grown at 30°C, 37°C and 45°C. T6P concentration was back calculated from a T6P standard curve and normalized to the input weight of fungal biomass. In all conditions, T6P accumulated in conidia and mycelium of the orlA mutant and was significantly higher than the wild-type (CEA10) (*P<0.05 in conidia, **P<0.05 in mycelia).

Fig. 7

mRNA abundance of trehalose phosphorylase genes (A) AFUB_062450 (B) AFUB_037080 and (C) orlA was measured in conidia from the wild-type (CEA10) and orlA mutant. RNA samples were extracted from conidia cultured at 30°C, 37°C and 45°C. mRNA abundance was normalized to actin and expression values are relative to the CEA10 sample at low temperature (30°C). Results are the mean and standard deviation of two biological replicates and were calculated using the 2^-ΔΔCt method (Livak & Schmittgen, 2001).

Fig. 8

Free inorganic phosphate (Pi) is sequestrated by T6P in the absence of OrlA. Conidia (A) and mycelia (B) biomass for Pi assays were cultured at 30°C, 37°C and 45°C. At all conditions, Pi levels found in mycelia of the orlA mutant were significantly decreased from the wild-type and reconstituted strains (P value < 0.05 as indicated) where as an insignificant difference (NS; P value >0.05) was found in conidia cultured at 37°C and 45°C. However, at 30°C, a significant increase in Pi levels was found in the orlA mutant relative to wild type and reconstituted strains.

Fig. 9

Key steps in glycolysis are altered in the absence of OrlA. (A) Hexokinase activity of the wild-type, the orlA mutant and the reconstituted strain indicates a decrease in hexokinase activity in the absence of orlA (P value = 0.04) relative to wild-type. Results presented are the mean and standard deviations from three independent experiments. (B) Pyruvate decarboxylase activity is decreased in the absence of OrlA also suggesting a potential decrease in glycolytic flux. Respective strains were grown in GMM at 37°C in normoxic (20% O₂) or hypoxic (1% O₂) conditions. Results presented are the mean and standard deviations from two independent experiments. *P = 0.03 **P = 0.05 (wild-type vs. mutant respectively for normoxia and hypoxia).

Fig. 10

OrlA is a critical component of the Aspergillus fumigatus virulence arsenal. (A) Outbred CD-1 male mice (n = 10 each fungal strain or mock control) were immunosuppressed as described in the experimental procedures to generate persistently neutropenic mice. Mice were inoculated intranasally with 10⁶ conidia/25 μl of wild-type CEA10, ΔorlA, or the orlA reconstituted strain Rec-orlA. A cohort of 10 mice was also mock infected with 0.01% Tween80 in sterile phosphate buffered saline. No mock infected animals perished in either experiment. Infection experiments were repeated two times with similar results. Log-Rank tests revealed that the survival curves between the mutant and wild-type CEA10 and mutant and reconstituted strain were statistically significant (P < 0.0001 and P = 0.0007, respectively). No significant difference was observed between the wild-type and reconstituted strains (P = 0.2659). (B) X-CGD murine model of IPA, mice were inoculated intranasally with 10⁵ conidia/25 μl of wild type and orlA mutant. The delay in death of mice infected with the orlA mutant is statistically significant (P value = 0.0005).

Fig. 11

Histopathology from immunosuppressed CD1 mouse model observed on day 3 after infection. Mice were intranasally inoculated with 10⁶ conidia and euthanized on day 3 post infection. Lung sections of representative infections stained with hematoxylin and eosin (H&E) or Gomori methenamine silver stain (GMS) are presented. Mock infected animals display normal healthy lung architecture with lack of necrosis, inflammation, and fungal elements. Wild-type (CEA10) and reconstituted strain infected lungs displayed significant numbers of inflammatory and necrotic lesions (H&E stains) surrounding sites infiltrated by fungal growth (black stained fungal hyphae as observed via GMS staining). Interestingly, the orlA mutant infected mice consistently contained less necrosis, inflammation, and fungal growth. Bar = 500 μm for 40× magnification.