Aspergillus fumigatus calcipressin CbpA is involved in hyphal growth and calcium homeostasis

Abstract

Calcineurin is a conserved protein phosphatase that plays a critical role in Ca(2+) signaling and stress responses. Previously, a new class of conserved calcineurin-binding proteins, the calcipressins, was identified. However, the role of these proteins remains controversial, and both inhibitory and stimulatory effects on calcineurin were observed. In this study, we investigate the role of CbpA, the Aspergillus fumigatus member of the calcipressin family, and report that deletion of the cbpA gene resulted in reduced hyphal growth and limited attenuated virulence. Interestingly, under high-calcium-level conditions, the DeltacbpA strain displayed improved Ca(2+) tolerance compared to the wild-type strain and revealed increased expression of vcxA, chsA, and cnaA, which encode the vacuolar Ca(2+)/H(+) exchanger VcxA, chitin synthase A, and the calcineurin catalytic subunit CnaA, respectively. The increased transcript levels of these three genes were reversed in the presence of the calcineurin inhibitor FK506, indicating a calcineurin-dependent mechanism. Overexpression of cbpA resulted in decreased transcription of vcxA, chsA, and cnaA, associated with wild-type sensitivity to Ca(2+). Taken together, our study highlights the importance of CbpA in the regulation of hyphal growth and calcium adaptation of A. fumigatus and provides evidence that CbpA may serve as a feedback inhibitor in some aspects of calcineurin functions.

FIG. 1.

Multiple alignment of Aspergillus fumigatus CbpA with predicted homologs from Homo sapiens, Cryptococcus neoformans, and Saccharomyces cerevisiae. Identical residues are in black, and similar amino acids are in gray. The most conserved central motif is underlined.

FIG. 2.

Replacement and complementation of the cbpA gene. (A). Schematic representation of the genomic locus of the wild-type and ΔcbpA strains. The cbpA gene was replaced with the pyrG gene by homologous recombination. (B) Complementation of cbpA resulting from a single crossover between the PacI site in the promoter region of the cbpA mutant allele and the PacI-linearized vector, containing the entire cbpA gene plus a 2-kb 5′ and 500-bp 3′ flanking sequence. (C) Southern analysis with EcoRV-digested genomic DNA and the cbpA left flank probe (P). The appearance of a 2.9-kb fragment in the ΔcbpA strain indicated successful gene replacement of cbpA by pyrG. The regeneration of the 3.5-kb wild-type (WT) EcoRV fragment and the appearance of a 7-kb EcoRV fragment comprising the pCB1636 plasmid sequence confirmed a successful reintroduction of cbpA into the ΔcbpA background at the native locus.

FIG. 3.

Hyphal growth analysis of the wild-type, ΔcbpA, and ΔcbpA+cbpA strains. (A) Culture morphology of the wild-type, ΔcbpA, and ΔcbpA+cbpA strains on GMM after 4 days of growth at 37°C. (B) The ΔcbpA strain displayed an approximately 30% decrease in radial growth over a 5-day time course compared with the wild-type and complemented strains.

FIG. 4.

Hyphal growth in the presence of CaCl₂. A total of 1 × 10⁴ conidia were inoculated onto GMM plates containing CaCl₂ (200 mM) with or without the calcineurin inhibitor FK506 (5 ng/ml). The radial growth was documented after 72 h at 37°C. The experiment was performed in triplicate and repeated three times with similar results. WT, wild type.

FIG. 5.

Generation of the pniiA-cbpA strain. (A) The promoter replacement construct comprises the hygromycin B resistance cassette, 1.2 kb upstream (L arm), 1.5 kb downstream from the predicted ATG start codon of the cbpA gene (R arm), and 1 kb of the nitrate-inducible promoter sequence (pniiA). (B) Southern analysis of SpeI-digested genomic DNA using the left flank probe (P) displayed an expected 4.8-kb fragment in the wild-type strain and a 7.2-kb fragment in the pniiA-cbpA mutant, confirming successful replacement of the cbpA endogenous promoter by the nitrate-inducible promoter pniiA.

FIG. 6.

CbpA plays a limited role in pathogenesis of A. fumigatus. (A) Kaplan-Meier survival curve. Infection with the wild-type strain resulted in 90% mortality by 14 days postinfection, while infection with the ΔcbpA mutant displayed a moderate reduction in mortality (P = 0.02). (B) Lung histopathology at day 7 postinfection. (Top row) Gomori's methenamine silver staining demonstrated extensive hyphal proliferation in the lung tissues of mice infected with the wild-type, ΔcbpA, and cbpA complemented strains. (Bottom row) Hematoxylin and eosin staining shows similar necrosis and inflammation levels in the three strains. Magnification, ×400.