Pneumocystis : A Polysaccharide Anomaly

Pneumocystis (Pc) is an opportunistic fungal pathogen that is well known for its ability to cause severe pneumonia in immunosuppressed individuals. The diagnosis of Pneumocystis pneumonia is commonly made by confirming the presence of the organism in specimens by staining with the fluorescent dye Calcofluor White, a non‐specific fluorochrome that is best known for staining fungal and plant cell walls by binding to β‐linked polysaccharides such as chitin and cellulose respectively. Previously, it was assumed that Calcofluor White, also known as Fluorescent Brightener 28 (FB28), was staining Pc by binding to chitin in Pc cyst form cell walls. However, recent genomic analysis of three species of Pc reveals that none of them encode the enzymes chitin synthase or chitinases needed for chitin synthesis and degradation, respectively. The absence of these genes means that Pc does not contain chitin in its cell walls. Therefore, we investigated what Calcofluor White is staining in Pneumocystis. First, Histochoice (Sigma Aldrich; St. Louis, MO)‐fixed, paraffin‐embedded (HCPE) Pc infected mouse lung sections were stained with FB28 (Sigma Aldrich; St. Louis, MO) at multiple concentrations and incubation times alone and in combination with Evans Blue (EB) (Fisher Scientific; Hampton, NH). Additionally, the Fungi‐Fluor Pneumocystis Kit (Polysciences; Warrington, PA), a one‐step detection kit for Pc containing FB28, was also tested. In order to test a different sample type and fixation process, homogenized infected mouse lung was used to create both unfixed cell smears and paraffin‐embedded, paraformaldehyde‐fixed Histogel (Richard‐Allan Scientific; San Diego, CA) sections. All samples were reviewed on a Nikon 90i epifluorescence microscope equipped with multiple filter sets enabling review of the tissues in the DAPI, FITC, TRITC and Cy5 channels. Regardless of concentration and incubation time, there was no staining of the Pc cysts in the heavily infected HCPE tissues in any channel. The Fungi‐Fluor kit also failed to stain the cysts in the HCPE sections as well as in the manufacturer provided control slides. It was suspected that the Histochoice fixation process played a role in the failure of these stains. The FB28, EB, FB28 and EB together, and Fungi‐Fluor stains were then applied to the cell smears and pellet preparations. Pc cysts fluoresced brightly and were easily visible in the cell smears in the expected DAPI channel as well as in the red and far red channels when EB was used. Cysts were less prominent but still visible in the cell pellet sections. Since Pc is known to contain the β‐linked polysaccharides β‐1,3‐ and β‐1,6‐glucan in its cell walls, we also examined if signal for β‐glucan co‐localized with FB28 staining. Dual labeling with biotinylated G Factor, a recently validated marker for β‐1,3‐glucan, visualized with Alexa Fluor 488 conjugated streptavidin, and FB28 staining was conducted to ascertain whether their signals co‐localized. Labeling with G Factor failed to co‐localize with the FB28 staining, strongly suggesting that β‐1,3‐glucan is not the target of the FB28 stain. For now, the target of FB28 stain in Pc cysts remains unknown. Support or Funding Information This research has been funded by Dr. Davis' startup funds from the Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University and the Capstone Grant from The Histochemical Society. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .