Precision-cut lung slices as an ex vivo model to study Pneumocystis murina survival and antimicrobial susceptibility

ABSTRACT Pneumocystis pneumonia (PCP) is a serious fungal lung infection caused by Pneumocystis jirovecii in immunosuppressed individuals. The lack of viable in vitro/ex vivo PCP models has greatly hindered the progress in studying the biology of these fungi, the host/pathogen interactions, as well as antifungal susceptibility testing. In this study, we show the utility of precision-cut lung slices (PCLS) to support the survival of Pneumocystis murina in vitro. We cultured PCLS tissue derived from wild type and immunocompromised mice with a P. murina inoculum in submerged or air-liquid interface models for up to 14 days. We isolated total RNA from the cultured lung tissues at days 0, 3, 7, 10, and 14 and analyzed for the expression of host lung genes and P. murina genes (Gsc1 for asci and Sp for trophs) by real-time quantitative PCR. When cultured in media alone, P. murina died gradually within a few days. However, when cultured on PCLS, both the troph and ascus forms survived throughout the incubation period of 2 weeks. Moreover, immunohistochemistry staining of P. murina inoculated PCLS sections using polyclonal anti-Pneumocystis sera and showed evidence of fungal aggregation and possible biofilm formation. Additionally, in vitro (PCLS) antibiotic susceptibility testing using commonly used antifungal drugs confirmed successful targeting of the troph and ascus forms by trimethoprim sulfamethoxazole and the ascus form by echinocandins. IMPORTANCE Our study reveals the potential of precision-cut lung slices as an ex vivo platform to study the growth/survival of Pneumocystis spp. that can facilitate the development of new anti-fungal drugs.

A total of 8 aliquots (120 µL each) of Pneumocystis murina inoculum (1X10 5 asci per mL) were cultured separately in air-liquid interface PCLS system as previously described under "PCLS culture".The cultures were harvested on day 14 of culture and P. murina organisms were isolated from the mixed cultures by centrifugation at 1500xg for 10 minutes at 4 o C. The resulting pellet was washed twice in 1 mL of 1x PBS by differential centrifugation and resuspended in 1 mL 1x PBS.The number of P. murina mitochondrial small subunit (mtSSU) ribosomal RNA copies per mL of suspension was approximately 4 x 10 3 by RT-qPCR.Four 6-weeks old male and female naïve Rag2 -/-Il2rγ -/-mice were challenged with 100 µL of the prepared inoculum via oral pharyngeal instillation as previously described [10,43].The mice were sacrificed four weeks post inoculation and total RNA was extracted from the homogenized right lung using Trizol reagent (ThermoFisher, 15596018) according to manufacturer's protocol.Subsequently, cDNA was synthesized from 1 µg [10, 44] of total RNA per 20µL reaction using the Bio-Rad iScript cDNA Synthesis Kit (1708841).The lung P. murina burdens were subsequently quantified by real-time PCR using SsoAdvanced Universal Probes Super mix (BIO-RAD, 1725281), with the following P. murina-specific mitochondrial small subunit (mtSSU) rRNA primers: Forward: 5′-5′-TCGGACTTGGATCTTTGCTTCCCA; Reverse: 5′-CATTCCGAGAACGAACGCAATCCT; and FAM probe: 5′-TCATGACCCTTATGGAGTGGGCTACA (all from IDT).Threshold cycle values were converted to RNA copy numbers using a premade standard of known Pneumocystis mtSSU rRNA as described previously standards were prepared as described previously [36].

RNA extraction and quantification
Total RNA was extracted from P. murina inoculated PCLS on day 3 and day 14 of culture using TRIzol LS reagent (ThermoFisher Scientific, 10296010) in accordance with the TRIzol LS Reagent manufacturer's protocol.The total RNA was DNase treated and quantified using the Qubit RNA HS assay kit (Thermo Fisher Scientific, Q32855).RNA quality was determined on an Agilent TapeStation 4150 using an Agilent RNA ScreenTape (Agilent, 5067-5576).

NEB total RNA Library Prep
About 10 ng of each total RNA sample was used to generate total RNA libraries using the NEBNext rRNA Depletion Kit v2 (Human/Mouse/rat) (NEB #E7400) and NEBNext UltraII Direction RNA Library Prep Kit for Illumina (NEB #7760).Subsequently, Final cDNA libraries containing NEB Dual Index (#E6440) were quantified using Qubit dsDNA HS assay kit (Thermo Fisher Scientific, Q32854).The quality of the libraries was determined by running each on an Agilent TapeStation 4150 using an Agilent D1000 ScreenTape (Agilent: 5067-5582).Smear analysis was performed using Agilent TapeStation Software (Version 4.1.1)with a range of 200-800bp to determine the average size of each library.The molarity of each library was then calculated based on size and concentration.All libraries were pooled at final concentration of 750pM with a spike-in of 2% PhiX control library v3 (Illumina, FC-110-3001).A mixture of pooled libraries was loaded on an Illumina NextSeq P2 (100) reagent cartridge (Illumina, 20046811).Single read and dual indexing sequence, 100 x 8 x 8, was performed on NextSeq2000, yielding approximately 66M Single reads per sample.Fastqs generated by Illumina BaseSpace DRAGEN Analysis Software (Version 1.2.1) were applied for further data analyses.The data generated was compared to RNA sequencing data from an in-vivo P. murina infection study.

Verification of viability of the cultured P. murina organisms Fig S1
Lung fungal burdens of Rag2 -/-Il2rγ -/-inoculated with P. murina cultured in the PCLS system for 14 days.(A) P. murina mtSSU rRNA copies standard curve.(B) P. murina mtSSU rRNA lung burdens of mice inoculated with cultured P. murina organisms.Each data point represents a mouse.

Table S1
Fig S2 RNA sequencing data Venn diagrams comparing the expression of Pneumocystis murina genes in PC-inoculated PCLS at day 3 and day 14 of culture with 14 day in-vivo P. murina infection study (GK1.5 treated/CD4 depleted mice).(A, B) Venn diagrams showing the common genes between (A) PC-inoculated PCLS at day 3 of culture and the 14 day in-vivo infection study, and (B) PC-inoculated PCLS at day 14 of culture and the in-vivo infection study.To generate this Venn diagram, day 3 inoculated and day 14 inoculated abundance were used from day 3 and day 14 of culture versus Naïve PCLS comparisons.For the in-vivo study, GK 1.5 abundance was used.Abundance threshold was set at >5 for all data curations.
Table of differentially expressed P. murina genes in P. murina inoculated PCLS at day 3 and day 14 of culture, from Cuffdiff.