Article Figures & Data
Figures
- FIG. 1.
A. fumigatus prtT encodes a conserved putative transcription factor of the Zn2Cys6 type. (A) The existence of the first annotated intron was examined by diagnostic PCRs covering the 5′ region to indicate that the prtT coding sequence is preceded by an unusually long leader sequence. The putative N terminus based on the putative intron is depicted in gray and the actual coding sequence in black. Priming oligonucleotides used are Sv589 (1), Sv576 (2), and AB51 (3), and calculated fragment sizes amplified from genomic DNA (g) or cDNA (c) are indicated in the left margin. A 100-bp DNA marker (M) was used as a size standard. Results were consistent between strain ATCC 46645 and an unrelated A. fumigatus isolate (D141 [data not shown]). The larger amplicon with primers Sv589 and AB51 (1/3) from cDNA is most likely due to contamination with genomic DNA. (B) Global alignment of the deduced A. fumigatus PrtT sequence with fungal orthologs from A. niger (A.n.; accession number XP_001402055), A. oryzae (A.o.; BAF74781), A. clavatus (A.c.; XP_001271888), and Penicillium crysogenum (P.c.; CAP98521). Consensus residues are boxed in black, and the gray bar indicates the position of the highly conserved Zn2Cys6 DNA binding domain.
- FIG. 2.
The prtT gene supports protein assimilation by A. fumigatus. (A) Deletion and reconstitution of the prtT locus. The genomic situation at the target locus is shown schematically for the wild-type progenitor ATCC 46645, the deletant AfS61, and the reconstituted strain AfS62. On the right side confirmative Southern analyses after digestion of genomic DNA with BspHI and EcoRV and hybridization with a 3′-specific probe (black bar) are displayed. (B) Growth tests on minimal medium supplemented with BSA with or without peptone as a nitrogen source. Media were point inoculated with equal amounts of spores and incubated at 37°C to reveal a severe growth retardation of the prtTΔ strain when undigested protein serves as the nitrogen source, a phenotype that is completely reversed when hydrolyzed protein such as peptone is added. (C) Growth on casein-containing medium shows the absence of a clearance halo around the growing edge of the AfS61 (prtTΔ) colony and therefore deficient degradation of precipitated protein. The inoculation legend presented on the right is valid for panels B and C.
- FIG. 3.
PrtT is required for expression of extracellular proteolytic activities. (A) Supernatants from cultures supplemented with various sources of nitrogen were spotted on an unprocessed X-ray film to test for the presence of proteolytic activities. In the presence of the primary N source ammonium, expression of secreted proteases that would hydrolyze the gelatin-containing light-sensitive layer is absent, but not in the presence of protein or the proteolysis product peptone. In contrast to the wild-type isolate (wt) and the reconstituted strain (rec.) the prtTΔ mutant apparently is impaired in producing extracellular proteases. Drop zones are indicated by dashed circles; untreated medium (m) was included in the spot tests. (B) Visualization of protein degradation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after 24 and 48 h of cultivation supports the near-complete absence of extracellular proteases in the culture supernatants containing BSA and peptone when inoculated with the prtTΔ deletion strain AfS61. (C) Quantitative assessment of extracellular proteolytic activities secreted by the wild-type isolate, the prtTΔ deletion strain and the reconstituted strain when grown in the presence of BSA and peptone reveals the almost-complete absence of extracellular proteases in the prtTΔ culture supernatant. Error bars indicate standard deviations.
- FIG. 4.
PrtT regulates transcriptional induction of several secreted proteases. Steady-state levels of transcripts from several protease-encoding genes were monitored by qRT-PCR after a shift from minimal medium containing NH4+ (N) as a nitrogen source to BSA (B)-containing cultures for 2 h. Expression rates were normalized to mRNA levels of the β-tubulin-encoding gene (AFUA_1G10910) and set arbitrarily to 1 for the wild-type strain grown in minimal medium, except for expression of dppIV, which could not be detected in either strain grown in NH4+-containing medium; in this case, the transcript level of the BSA-grown wild-type strain was set arbitrarily to 100. Error bars indicate standard deviations.
- FIG. 5.
The prtTΔ deletant strain grows at rates comparable to those for the wild-type reference in the lungs of leukopenic mice. Shown are weight loss data for infected animals (A) and threshold cycle (Ct) values from qPCRs performed on DNA samples extracted from corresponding lung tissues (B). Similar disease progressions and quantities of fungal burden can be deduced from these data, which indicates that the deletion mutant AfS61 is not impaired in virulence in this kind of disease model.
Tables
- TABLE 1.
Fungal strains and plasmid constructs
Strain or plasmid Description Reference or source Strains ATCC 46645 Aspergillus fumigatus wild-type strain 13 AfS61 prtTΔ deletion mutant (prtT::loxP-Phleor-loxP) This study AfS62 Reconstituted strain (prtT<ptrA>) This study Plasmids pJET1.2 Positive selection vector, pUC19 derivative (bla, multiple-cloning site in Eco47IR) Fermentas pSK275 Plasmid containing ptrA cassette conferring resistance to pyrithiamine 27 pSK341 Aspergillus marker cassette conferring resistance to phleomycin (pgpdA::ble/tk::trpCt) 26 pSK462 Replacement cassette for deletion of prtT in A. fumigatus This study pSK463 Reconstitution cassette containing prtT gene coupled to ptrA resistance cassette This study - TABLE 2.
Oligonucleotides used in this study
Oligonucleotide Sequence AB17 5′-TAT AGT TTA AAC ATT AGG TAA ATG CAT TCT AC-3′ AB18 5′-TAT AGT TTA AAC AAG AAT TGA CTG ATA TGC TA-3′ AB19 5′-TAT AGG CCA TCT AGG CCT ATT GTC AAT CAT ACA TGG AAT T-3′ AB20 5′-TAT AGG CCT GAG TGG CCG GCG GTT GAG TCT TAA ATT TTC-3′ AB28 5′-CCA ATG ACA GCA GTG TGT CC-3′ AB29 5′-TCC CCG TCC AAG CTG TAG C-3′ AB30 5′-CAA ACT CAC GAG GAG ATG AAG-3′ AB31 5′-GCA CTG AGG GGT GAT GAC C-3′ AB34 5′-TGC CTA CGT TGT CGA CAG TG-3′ AB35 5′-TTT GAG GCA TCA CTG TTC TCG-3′ AB36 5′-TGA CTC GGA ATA CCT GAC CC-3′ AB37 5′-CCA ACT GGG ACT TGA CAG TG-3′ AB38 5′-ACT TTC CGC GTG GTC GAC G-3′ AB39 5′-GTC GGT CTC AAC TCT CCA TG-3′ AB51 5′-GTT CCA GCG AGA TGA GGC G-3′ Sv551 5′-GGG AGA TCT GAC AGA CGG GCA ATT G-3′ Sv556 5′-GAT GAG ATC TTG CAT CTT TGT TTG TAT TAT AC-3′ Sv576 5′-GTC ATT TGT CGG CTG CTG ATC C-3′ Sv589 5′-ACA GAG GCC CAT CGA TAT CGC TGC-3′ TubFw 5′-GGA CGT TAC CTC ACC TGC TC-3′ TubRev 5′-CAC GCT TGA ACA ACT CCT GA-3′ RTDpp4Fw 5′-TGT CGA CGC ACA GCA CAT CG-3′ RTDpp4Rev 5′-CCG GAT CGC ACT GGC ATT GT-3′ qPCRf 5′-GAT ACC GTC GTA GTC TTA-3′ qPCRr 5′-TGT CTG GAC CTG GTG AGT-3′
