Pneumocystis jirovecii Rtt109, a Novel Drug Target for Pneumocystis Pneumonia in Immunosuppressed Humans

Abstract

f 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.

Rtt109‐dependent histone H3 K56 acetylation and gene activity are essential for the biological control potential of Beauveria bassiana

“…Rtt109 was proven to localize in the nuclei of hyphal cells and specifically to catalyze histone H3K56 acetylation in B. bassiana . This is consistent with the same site‐specific activity of its orthologues in yeasts and yeast‐like fungi, and highlights a tight link of Rtt109 to the histone chaperone Asf1 in B. bassiana and conserved activity of Rtt109 for the link in both yeasts and filamentous fungi. H3K56 acetylation allows for deposition of newly synthesized H3 molecules into the genome during DNA replication and chromatin assembly after DNA damage repair and hence is essential for genomic stability, DNA damage repair, DNA replication and global gene activity .…”

“…Loss‐of‐function mutation of Rtt109 blocks formation of white and opaque cell types and pathogenesis in C. albicans . In the yeast‐like fungi Pneumocystis spp., the Rtt109 orthologue also catalyzes the H3K56 acetylation required for genotoxic resistance, DNA replication and DNA damage repair, and the catalytic activity is associated with Asf1 . Previous studies demonstrate that Rtt019 acetylates the specific site H3K56 when associated with Asf1 but other K56‐exclusive sites on H3 when associated with Vps75.…”

Characterization of N-Acetylglucosamine Biosynthesis in Pneumocystis species. A New Potential Target for Therapy

“…Procedures in the supplement include: immunofluorescence detection of GlcNAc reactivity in Pc (19)(20)(21)(22)(23)(24)(25)(26), Western analysis of Pc GlcNAc complexed to proteins (27,28); verification of GlcNAc biosynthetic genes in the Pc and Pneumocystis jirovecii (Pj) genomes (16); expression of Pmuap1 mRNA in Pm-infected mice (29); expression, purification, and enzymatic activity of PmUap1 (2,16,(30)(31)(32); determination of Pc synthesis of GlcNAc (33)(34)(35); glycosyl composition of Pc GlcNAc preparations using gas chromatography/mass spectrometry (GC/MS) (36)(37)(38)(39); and nikkomycin Z treatment of Pc-infected rats (40)(41)(42). Furthermore, all nucleotide sequence accession numbers are included in the supplement.…”

“…For these studies, Pc and Pm organisms were originally derived from American Type Culture Collection (Manassas, VA) stocks and propagated in corticosteroid-treated rats or mice, as reported previously (16,17). Populations of Pc were isolated from chronically infected rat lungs by homogenization and filtration through 10-mm filters, as we previously described (3,18).…”

Post-HTS case report and structural alert: Promiscuous 4-aroyl-1,5-disubstituted-3-hydroxy-2 H -pyrrol-2-one actives verified by ALARM NMR

“…6 Importantly, there is no known mammalian Rtt109 homolog, which has led to the hypothesis that selective, potent inhibitors of Rtt109-catalyzed histone acetylation can function as minimally-toxic antifungal agents. 7–10 …”