To increase the frequency of homologous recombination, we inactivated the KU80 homologue in Aspergillus fumigatus (named akuB KU80 ). Homologous integration reached about 80% for both calcineurin A (calA) and polyketide synthase pksP (alb1) genes in the akuB KU80 mutant to 3 and 5%, respectively, when using a wild-type A. fumigatus strain. Deletion of akuB KU80 had no influence on pathogenicity in a low-dose murine infection model. Double-strand breaks are the most severe form of DNA damage. Eukaryotes have two main pathways to deal with this type of DNA damage: (i) homologous recombination, involving interaction between homologous sequences, and (ii) nonhomologous end joining, involving direct ligation of the strand ends independently of DNA homology (17). The most important double-strand break repair pathway in Saccharomyces cerevisiae is homologous recombination (18,20), while other organisms, such as humans, preferentially use nonhomologous end joining. The nonhomologous end-joining process is mediated by the DNA-dependent protein kinase catalytic subunit, the Ku70-Ku80 heterodimer, and the DNA ligase IV-Xrcc4 complex (17). Recently, Ninomiya et al. (13) disrupted Neurospora crassa genes homologous to human KU70 and KU80.
SummaryThe protein phosphatase calcineurin is an important mediator connecting calcium-dependent signalling to various cellular responses in multiple organisms. In fungi calcineurin acts largely through regulating Crz1p-like transcription factors. Here we characterize an Aspergillus fumigatus CRZ1 homologue, CrzA and demonstrate its mediation of cellular tolerance to increased concentrations of calcium and manganese. In addition to acute sensitivitiy to these ions, and decreased conidiation, the crzA null mutant suffers altered expression of calcium transporter mRNAs under high concentrations of calcium, and loss of virulence when compared with the corresponding complemented and wild-type strains. We use multiple expression analyses to probe the transcriptional basis of A. fumigatus calcium tolerance identifying several genes having calA and/or crzA dependent mRNA accumulation patterns. We also demonstrate that contrary to previous findings, the gene encoding the Aspergillus nidulans calcineurin subunit homologue, cnaA, is not essential and that the cnaA deletion mutant shares the morphological phenotypes observed in the corresponding A. fumigatus mutant, DcalA. Exploiting the A. nidulans model system, we have linked calcineurin activity with asexual developmental induction, finding that CrzA supports appropriate developmental induction in a calcineurin and brlA-dependent manner in both species.
Invasive aspergillosis is predominantly caused by Aspergillus fumigatus, and adaptations to stresses experienced within the human host are a prerequisite for the survival and virulence strategies of the pathogen. The central signal transduction pathway operating during hyperosmotic stress is the high osmolarity glycerol mitogen-activated protein kinase cascade. A. fumigatus MpkC and SakA, orthologues of the Saccharomyces cerevisiae Hog1p, constitute the primary regulator of the hyperosmotic stress response. We compared A. fumigatus wild-type transcriptional response to osmotic stress with the ΔmpkC, ΔsakA, and ΔmpkC ΔsakA strains. Our results strongly indicate that MpkC and SakA have independent and collaborative functions during the transcriptional response to transient osmotic stress. We have identified and characterized null mutants for four A. fumigatus basic leucine zipper proteins transcription factors. The atfA and atfB have comparable expression levels with the wild-type in ΔmpkC but are repressed in ΔsakA and ΔmpkC ΔsakA post-osmotic stress. The atfC and atfD have reduced expression levels in all mutants post-osmotic stress. The atfA-D null mutants displayed several phenotypes related to osmotic, oxidative, and cell wall stresses. The ΔatfA and ΔatfB were shown to be avirulent and to have attenuated virulence, respectively, in both Galleria mellonella and a neutropenic murine model of invasive pulmonary aspergillosis.
This study evaluated the morphological changes produced by LLLT on the initial stages of bone healing and also studied the pathways that stimulate the expression of genes related to bone cell proliferation and differentiation. One hundred Wistar rats were divided into control and treated groups. Noncritical size bone defects were surgically created at the upper third of the tibia. Laser irradiation (Ga-Al-As laser 830 nm, 30 mW, 94 s, 2.8 J) was performed for 1, 2, 3, 5, and 7 sessions. Histopathology revealed that treated animals produced increased amount of newly formed bone at the site of the injury. Moreover, microarray analysis evidenced that LLLT produced a significant increase in the expression TGF-β, BMP, FGF, and RUNX-2 that could stimulate osteoblast proliferation and differentiation, which may be related to improving the deposition of newly formed bone at the site of the injury. Thus, it is possible to conclude that LLLT improves bone healing by producing a significant increase in the expression of osteogenic genes.
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