Tools for Assessing Translation in Cryptococcus neoformans

Abstract

Cryptococcus neoformans is a ubiquitous environmental fungus capable of establishing an infection in a human host. Rapid changes in environments and exposure to the host immune system results in a significant amount of cellular stress, which is effectively combated at the level of translatome reprogramming. Repression of translation following stress allows for the specific reallocation of limited resources. Understanding the mechanisms involved in regulating translation in C. neoformans during host infection is critical in the development of new antifungal drugs. In this review, we discuss the main tools available for assessing changes in translation state and translational output during cellular stress.

Keywords: elongation; host adaptation; initiation; repression; translation; translation regulation.

Figure 1

A schematic of a 43S preinitiation complex (the 40S small ribosomal subunit with eIF2 bound to GTP, and Met-tRNA^Met) loaded onto an mRNA, bound by eIF4F (composed of the cap binding protein eIF4E, the helicase eIF4A, and the scaffolding protein eIF4G). The scaffolding protein eIF4G spans to bind the poly-A binding protein, Pab1, resulting in circularization of the translational complex.

Figure 2

Polysome profiling and analysis. (A) Representative polysome traces for an unstressed C. neoformans culture (30° C) in blue, compared to a polysome profile subjected to temperature stress (37 °C) for 30 min. The decrease in area under both the polysome fraction and 80S monosome peak, along with an increase in the 60S subunit peak, is indicative of repressed translation, reduced initiation, and an increase in the free ribosome pool. (B) RNA following electrophoretic separation from isolated individual fractions from representative polysome profiles. Visible are fractions containing only 18S rRNA representing the small 40S ribosomal subunit, fractions with predominantly 28S rRNA representing the large 60S ribosomal subunits, a fraction containing a strong enrichment of 80S monosomes, and the polysome portion showing distribution of 80S ribosomes.

Figure 3

Measuring translational output. (A) Top panel: A western blot of whole lysates from representative samples treated with temperature stress for the indicated time, using an α-puromycin antibody to recognize puromycilated peptides, indicative of translational output. Bottom panel: Total protein for normalization of the α-puromycin signal. (B) A histogram of flow cytometry results following the click chemistry labeling reaction of incorporated HPG. The histogram in green shows basal levels of translational output while the histogram n red demonstrates inhibition of translational output by cycloheximide (CHX) treatment.