The human fungal pathogen Cryptococcus neoformans relies on a complex signaling network for the adaptation and survival at the host temperature. Protein phosphatase calcineurin is central to proliferation at 37°C but its exact contributions remain ill-defined. To better define genetic contributions to the C. neoformans temperature tolerance, 4031 gene knockouts were screened for genes essential at 37°C and under conditions that keep calcineurin inactive. Identified 83 candidate strains, potentially sensitive to 37°C, were subsequently subject to technologically simple yet robust assay, in which cells are exposed to a temperature gradient. This has resulted in identification of 46 genes contributing to the maximum temperature at which C. neoformans can proliferate (Tmax). The 46 mutants, characterized by a range of Tmax on drug-free media, were further assessed for Tmax under conditions that inhibit calcineurin, which led to identification of several previously uncharacterized knockouts exhibiting synthetic interaction with the inhibition of calcineurin. A mutant that lacked septin Cdc11 was among those with the lowest Tmax and failed to proliferate in the absence of calcineurin activity. To further define connections with calcineurin and the role for septins in high temperature growth, the 46 mutants were tested for cell morphology at 37°C and growth in the presence of agents disrupting cell wall and cell membrane. Mutants sensitive to calcineurin inhibition were tested for synthetic lethal interaction with deletion of the septin-encoding CDC12 and the localization of the septin Cdc3-mCherry. The analysis described here pointed to previously uncharacterized genes that were missed in standard growth assays indicating that the temperature gradient assay is a valuable complementary tool for elucidating the genetic basis of temperature range at which microorganisms proliferate.

Objective: Split-hand/foot malformation (SHFM) is a rare, often debilitating, congenital limb malformation. A single nucleotide polymorphism within the leucine zipper containing kinase AZK (ZAK) gene was recently associated with SHFM in two consanguineous Pakistani pedigrees. We hypothesized that additional unrelated patients with the phenotype may carry a pathogenic mutation in ZAK. Methods: DNA samples were collected from 38 patients with SHFM and associated hearing loss for Sanger DNA sequencing and in silico analysis. Results: Two missense mutations within ZAK were detected in 11 patients, but only one missense variant, p.Ala505Ser, occurred with a presumed rare allele frequency. In silico modeling of the ZAK protein with the p.Ala505Ser substitution indicated a negative binding free energy change (mean