Deciphering cell wall integrity signalling in Aspergillus fumigatus: identification and functional characterization of cell wall stress sensors and relevant Rho GTPases.

The fungal cell wall, a conserved and highly dynamic structure, is essential for virulence and viability of fungal pathogens and is an important antifungal drug target. The cell wall integrity (CWI) signalling pathway regulates shape and biosynthesis of the cell wall. In this work we identified, localized and functionally characterized four putative CWI stress sensors of Aspergillus fumigatus, an airborne opportunistic human pathogen and the cause of invasive aspergillosis. We show that Wsc1 is specifically required for resistance to echinocandin antifungals. MidA is specifically required for elevated temperature tolerance and resistance to the cell wall perturbing agents congo red and calcofluor white. Wsc1, Wsc3 and MidA additionally have overlapping functions and are redundantly required for radial growth and conidiation. We have also analysed the roles of three Rho GTPases that have been implicated in CWI signalling in other fungi. We show that Rho1 is essential and that conditional downregulation of rho1 or deletion of rho2 or rho4 results in severely impaired CWI. Our data indicate significant functional differences between the CWI stress sensors of yeasts and moulds.

Prevalence of labyrinthine ossification in CT and MR imaging of patients with acute deafness to severe sensorineural hearing loss.

OBJECTIVE: To evaluate the prevalence of labyrinthine ossification, and especially cochlear ossification, in a cohort of patients with unilateral sudden deafness or severe sensorineural hearing loss. DESIGN: Retrospective data collection. STUDY SAMPLE: Sixty-four consecutive patients with unilateral sudden deafness or severe sensorineural hearing loss and either high-resolution CT (HRCT) of the temporal bone (isotropic spatial resolution ≤ 0.8 mm; n = 18) or high resolution CISS MRI (isotropic spatial resolution ≤ 1 mm; n = 55) were included. Nine patients underwent both imaging modalities. A standardized reading regarding labyrinthine ossifications was performed by an experienced head and neck radiologist blinded to clinical symptoms. RESULTS: Radiologic signs of cochlear ossification were present in 14 patients (12 CT and 2 MRI). Eight patients showed unilateral and six patients bilateral signs of cochlear ossification. In all except one of the unilateral cases, the deafened ear was affected. CONCLUSIONS: Signs of cochlear ossification were found in an unexpectedly high rate (14/64, 22%) of patients with acute deafness. The data suggest HRCT of the temporal bone to be more sensitive to detect labyrinthine ossification than MRI. HRCT of the temporal bone should therefore be considered in patients with impaired recovery of acute deafness to exclude cochlear ossification; if present, and, in cases of early signs, the patient should be evaluated further to facilitate early cochlear implantation before progression impedes electrode insertion, reflecting latest developments considering cochlea implants for single-sided deafness to be effective.

Farnesol misplaces tip-localized Rho proteins and inhibits cell wall integrity signalling in Aspergillus fumigatus.

Farnesol is known for inducing apoptosis in some fungi and mammalian cells. To evaluate its potential role as an antifungal agent, we studied its impact on the human pathogen Aspergillus fumigatus. We found that growth of A. fumigatus wild type is inhibited, but two cell wall mutants, Deltamnt1 andDeltaglfA, are much more susceptible to farnesol. This susceptibility is partially rescued by osmotic stabilization, suggesting that farnesol is a cell wall perturbing agent. However, farnesol does not activate but inhibit the cell wall integrity (CWI) pathway. Remarkably, mutants lacking AfMkk2 or AfMpkA, two kinases essential for CWI signalling, are also highly susceptible to farnesol, suggesting that its mode of action goes beyond inhibition of CWI signalling. Farnesyl derivatives are known for interfering with the function of prenylated proteins. We analysed the subcellular localization of two prenylated Rho family GTPases, AfRho1 and AfRho3, which are implicated in controlling CWI and the cytoskeleton. We found that under normal growth conditions AfRho1 and AfRho3 predominantly localize to the hyphal tip. After farnesol treatment this localization is rapidly lost, which is accompanied by swelling of the hyphal tips. Parallel displacement of tropomyosin from the tips suggests a concomitant disorganization of the apical actin cytoskeleton.

AfMkk2 is required for cell wall integrity signaling, adhesion, and full virulence of the human pathogen Aspergillus fumigatus.

The cell wall integrity (CWI) pathway, best characterized in S. cerevisiae, is strikingly conserved in Aspergillus species. We analyzed the importance of AfMkk2, a CWI signaling kinase, for virulence and antifungal therapy in the human pathogen A. fumigatus. A mutant lacking AfMkk2 is less adherent to glass and plastic surfaces and shows increased sensitivity to alkaline pH stress and antifungals. Rather than AfMpkA, the target kinase of AfMkk2, AfMpkB is activated in the mutant under cell wall stress. Interestingly, the mutant lacking AfMkk2 shows an enhanced sensitivity to posaconazole and voriconazole. And in agreement with its sensitivity to moderate temperatures, it is less virulent in a murine infection model. Our data underline the importance of mkk2 for the fitness, but also for the pathogenicity of A. fumigatus.

Value of routine plain x-ray position checks after cochlear implantation.

OBJECTIVE: To analyze the value of a routine x-ray position check after cochlear implantation and to assess if an increased resistance during electrode insertion is a sufficient predictor of electrode misplacement. STUDY DESIGN: Retrospective data collection. SETTING: University hospital. METHODS: Plain x-rays (Stenvers' projection) and the respective surgery reports of 218 patients having received cochlear implantation (243 ears) were analyzed for possible electrode misplacements and intraoperative conspicuities during electrode insertion. RESULTS: Electrode misplacement (tip-over, loop, kinking, scalar transition, and incomplete insertion) was observed in 8% of the entire study cohort, but only in 5% if cases with inner ear dysplasia or labyrinthine ossification (n = 28) were excluded from analysis. Intraoperatively, an increased resistance during electrode insertion was found in 16% but only in 8% when cases with inner ear dysplasia or labyrinthine ossification were excluded. The intraoperative finding of an increased resistance during electrode insertion had a sensitivity of 55% and a specificity of 88% for predicting radiographically confirmed electrode misplacements (positive predictive value, 29%; negative predicting value, 96%). CONCLUSION: Nearly half of the cases of electrode misplacement would have been overlooked if radiographic position checks would have been done only in patients with intraoperative conspicuities during electrode insertion. This finding advocates routine radiographic position checks, although electrode misplacements are relatively rare in patients with regular inner ear anatomy.