Protocol to evaluate translation inhibition in Candida species using fluorescence microscopy and flow cytometry

Abstract

Translation inhibitors have therapeutic potential against Candida species. Here, we present a protocol to measure translation inhibition in Candida spp. We describe steps for employing an alkynylated methionine analog, L-homopropargylglycine (HPG), that becomes incorporated into newly synthesized proteins. We then detail procedures to perform a click reaction of the alkyne with a fluorescent azide, which is visualized using fluorescence microscopy and quantified by flow cytometry. For complete details on the use and execution of this protocol, please refer to Puumala et al.,¹ Fu et al.,² and Iyer et al.³.

Keywords: genetics; microbiology; microscopy.

Graphical abstract

Figure 1

CytExpert “Acquisition” tab functions required for completion of this assay Display of Beckman Coulter CytExpert 2.3 software on the “Acquisition” tab used for collection of flow cytometry data. Legend highlights key selections required for the completion of the assay described. Ensure that all listed parameters have been selected prior to selecting “Record” to acquire data from each test sample.

Figure 2

CytExpert “Analyze” tab functions required for completion of this assay Display of Beckman Coulter CytExpert 2.3 software on the “Analyze” tab used for evaluation and plotting of flow cytometry data. Legend highlights key selections required for the completion of the assay described. Samples can be dragged from the left-side “Tube” menu bar onto the scatter (blue) and histogram (purple) graphs, which will automatically populate each plot as well as the statistics table (pink).

Figure 3

The use of flow cytometry to quantify translation inhibitory activity in C. albicans (A) Pseudo-colored density plot shows side-scatter area (SSC-A) vs. forward-scatter area (FSC-A) for each event acquired during analysis of a DMSO-treated sample of C. albicans (CaSS1) using a CytoFlex flow cytometer and CytExpert Software. An ellipsoid gating region (P1, red) encompassing the bulk population of events captured during the run is indicated. This region gates for events demonstrating the low scatter parameters characteristic of single cells while excluding debris (very low scatter events) and high scatter events representing doublets and aggregates of cells. Only gated events are used when plotting and analyzing the data displayed in (B and C). (B) To quantify fluorescent signal emitted by samples analyzed for inhibition of translation, cell suspensions prepared in step 15 were run on a CytoFlex flow cytometer and analyzed using CytExpert. Frequency histogram displays the distribution of relative fluorescence signal intensity (FITC-A filter; excitation: 488 nm, emission: 525/40 nm) for each sample population gated within P1, where higher FITC-A corresponds to increased levels of active translation. (C) Table summarizing the data collected by CytExpert upon analysis of samples plotted in B. Fold-change in median FITC-A was calculated, comparing each sample treatment to the DMSO solvent.

Figure 4

Fluorescence microscopy provides a complimentary approach to visualize translation inhibitory activity in C. albicans using the Click-iT HPG Alexa Fluor assay system C. albicans was treated with DMSO, DMSO in the absence of HPG, 2 μg/mL fluconazole (FLC), or 50 μM NP-BTA, and processed through all steps reported in the protocol. Fixed cells were imaged using a Zeiss Axio Imager.MI. Images were taken at 100x magnification using a Plan-Apochromat 100x/1,4 Oil lens (scale bar: 20 μm) under differential interference contrast (DIC) and an EGFP fluorescence filter (excitation: 488 nm, emission: 509 nm, 50 ms exposure). Fluorescent light source: X-Cite SERIES 120. GFP exposure time: 200 ms. DIC light source: TL halogen lamp. DIC exposure time: 52.6 ms.