Pneumocystis jirovecii Rtt109, a novel drug target for Pneumocystis pneumonia in immunosuppressed humans

Abstract

Pneumocystis pneumonia (PcP) is a significant cause of morbidity and mortality in immunocompromised patients. In humans, PcP is caused by the opportunistic fungal species Pneumocystis jirovecii. Progress in Pneumocystis research has been hampered by a lack of viable in vitro culture methods, which limits laboratory access to human-derived organisms for drug testing. Consequently, most basic drug discovery research for P. jirovecii is performed using related surrogate organisms such as Pneumocystis carinii, which is derived from immunosuppressed rodents. While these studies provide useful insights, important questions arise about interspecies variations and the relative utility of identified anti-Pneumocystis agents against human P. jirovecii. Our recent work has identified the histone acetyltransferase (HAT) Rtt109 in P. carinii (i.e., PcRtt109) as a potential therapeutic target for PcP, since Rtt109 HATs are widely conserved in fungi but are absent in humans. To further address the potential utility of this target in human disease, we now demonstrate the presence of a functional Rtt109 orthologue in the clinically relevant fungal pathogen P. jirovecii (i.e., PjRtt109). In a fashion similar to that of Pcrtt109, Pjrtt109 restores H3K56 acetylation and genotoxic resistance in rtt109-null yeast. Recombinant PjRtt109 is an active HAT in vitro, with activity comparable to that of PcRtt109 and yeast Rtt109. PjRtt109 HAT activity is also enhanced by the histone chaperone Asf1 in vitro. PjRtt109 and PcRtt109 showed similar low micromolar sensitivities to two reported small-molecule HAT inhibitors in vitro. Together, these results demonstrate that PjRtt109 is a functional Rtt109 HAT, and they support the development of anti-Pneumocystis agents directed at Rtt109-catalyzed histone acetylation as a novel therapeutic target for human PcP.

FIG 1

PjRtt109 is an orthologue of PcRtt109 and is homologous to yeast and other fungal Rtt109 proteins involved in H3K56 acetylation. (Top) Pairwise sequence alignment of PjRtt109 and PcRtt109. (Bottom) Multiple-sequence alignment of fungal Rtt109 histone acetyltransferases. Alignments were performed using default CLUSTALW settings (MacVector 12.5.1). PNJIR, Pneumocystis jirovecii; PNCAR, Pneumocystis carinii; SCPOM, Schizosaccharomyces pombe; SACER, Saccharomyces cerevisiae; CAALB, Candida albicans. Dark shading indicates identical residues, and light shading indicates similar residues; asterisks denote similar residues in the consensus sequence.

FIG 2

Pjrtt109 primer set amplifies a specific amplicon from P. jirovecii (Pj) genomic DNA but not human genomic DNA. hGAPDH, human glyceraldehyde-3-phosphate dehydrogenase.

FIG 3

Heterologous expression of Pjrtt109 restores H3K56ac levels and genotoxic resistance in rtt109-null yeast. (A) Complementation of rtt109-null yeast with Pjrtt109 restores H3K56ac, as assessed by Western blots of yeast whole-cell extracts. WT, wild-type strain plus control vector; Δ, rtt109-null strain plus control vector; PJ, rtt109-null strain plus Pjrtt109 cDNA. (B) Complementation of rtt109-null yeast with Pjrtt109 restores genotoxic resistance, as assessed by the growth of yeast on solid medium. Tenfold serial dilutions of S. cerevisiae were spotted on minimal medium (with 2% galactose minus uracil [−URA]) alone or with the addition of MMS, CPT, or HU.

FIG 4

PjRtt109 is an active HAT in vitro. (A) PjRtt109, expressed as a GST-fusion protein, shows in vitro HAT activity comparable to that of PcRtt109. ***, P < 0.001, compared with the REGα negative control. Shown are representative results from a single experiment, with similar results being obtained in at least two other independent experiments. (B) Reaction mixture aliquots, as shown in panel A, were resolved by SDS-PAGE and stained with CBB to demonstrate equal substrate and enzyme contents. Autoradiographs (AR) reveal that Rtt109-catalyzed histone acetylation is detected only on H3 and not on H4. (C) Western blot analysis of reaction mixture aliquots shows that PjRtt109, like PcRtt109, catalyzes H3K56ac in vitro. Equal substrate contents were verified with Ponceau S staining and Western blotting for H3. (D) PjRtt109 and SpRtt109 have similar HAT activities in vitro. (E) SDS-PAGE and CBB staining of reaction mixture aliquots, as shown in panel D, show equal protein contents. Autoradiographs show that PjRtt109 and PcRtt109 similarly catalyze the acetylation of H3, and H4 acetylation was not detected. (F) PjRtt109 and SpRtt109 both catalyze H3K56ac in vitro, as assessed by Western blotting. Equal substrate contents were verified with Ponceau S staining and anti-H3 Western blotting. (G) An extended-exposure autoradiograph using reaction mixture aliquots as shown in panel A demonstrates low levels of PjRtt109 autoacetylation (arrow).

FIG 5

PjRtt109 HAT activity is enhanced by the histone chaperone Asf1 in vitro. (A) PjRtt109 HAT activity in vitro is enhanced by the addition of ScAsf1. ***, P < 0.001, pairwise comparisons between the dH3–H4 and ScAsf1-dH3–H4 substrates for each enzyme or pairwise comparisons with the REGα negative controls. Shown are representative results from a single experiment, with similar results being obtained in at least two other independent experiments. (B) Autoradiography of reaction mixture aliquots, as shown in panel A, demonstrates that acetylation is detected only on H3 and not on H4 or ScAsf1. Equal substrate histone contents were verified by CBB staining. (C) Western blotting of reaction mixture aliquots analogous to those shown in panel B versus H3K56ac, using nonradiolabeled acetyl-CoA as the substrate, was performed. Equal histone substrate contents were verified by Ponceau S staining and Western blotting.

FIG 6

Reported small-molecule HAT inhibitors can inhibit PjRtt109 activity in vitro. (A) Inhibition of PjRtt109 activity by the reported HAT inhibitors garcinol (IC₅₀, 5.9 μM) and compound 1 (IC₅₀, 1.7 μM). Shown are representative results from a single experiment. (B) Comparison of IC₅₀s for the compounds from panel A versus PjRtt109 and PcRtt109, tested under similar conditions. Values represent the average and standard deviation of three independent experiments. (C) Autoradiography of reaction mixture aliquots from panel A, confirming dose-dependent inhibition by garcinol of PjRtt109-catalyzed histone acetylation in vitro. Equal histone substrate contents were verified by CBB staining. (D) Western blot confirmation of dose-dependent inhibition by garcinol of PjRtt019-catalyzed H3K56ac and H3K27ac in vitro. Reaction mixture aliquots are analogous to those shown in panel C except that nonradiolabeled acetyl-CoA was used as the substrate. Equal histone substrate contents were verified by Ponceau S staining (representative membrane shown).

FIG 7

Effects of HAT inhibitors on Pneumocystis viability. Freshly isolated P. carinii organisms were maintained ex vivo for 72 h in test medium, and organism viability was determined by relative ATP contents. Ampicillin (10 μg/ml) and pentamidine (1 μl/ml) were included as relevant negative- and positive-control compounds. The reported pan-HAT inhibitor garcinol exhibited a significant dose-dependent reduction in P. carinii viability. In contrast, the recently described Rtt109 inhibitor compound 1 failed to alter P. carinii viability under the conditions tested. A DMSO diluent control was also used. Shown are the results of three independent experiments. *, P < 0.05, compared with control ATP levels without test agent.