Characterization of the Pneumocystis carinii Histone Acetyltransferase Chaperone Proteins PcAsf1 and PcVps75

Am J Respir Cell Mol Biol

Hebrink

Jenson

et al. 2017

Self Cite

N-acetylglucosamine (GlcNAc) serves as an essential structural sugar on the cell surface of organisms. For example, GlcNAc is a major component of bacterial peptidoglycan, it is an important building block of fungal cell walls, including a major constituent of chitin and mannoproteins, and it is also required for extracellular matrix generation by animal cells. Herein, we provide evidence for a uridine diphospho (UDP)-GlcNAc pathway in Pneumocystis species. Using an in silico search of the Pneumocystis jirovecii and P. murina (Pm) genomic databases, we determined the presence of at least four proteins implicated in the Saccharomyces cerevisiae UDP-GlcNAc biosynthetic pathway. These genes, termed GFA1, GNA1, AGM1, and UDP-GlcNAc pyrophosphorylase (UAP1), were either confirmed to be present in the Pneumocystis genomes by PCR, or, in the case of Pm uap1 (Pmuap1), functionally confirmed by direct enzymatic activity assay. Expression analysis using quantitative PCR of Pneumocystis pneumonia in mice demonstrated abundant expression of the Pm uap1 transcript. A GlcNAc-binding recombinant protein and a novel GlcNAc-binding immune detection method both verified the presence of GlcNAc in P. carinii (Pc) lysates. Studies of Pc cell wall fractions using highperformance gas chromatography/mass spectrometry documented the presence of GlcNAc glycosyl residues. Pc was shown to synthesize GlcNAc in vitro. The competitive UDP-GlcNAc substrate synthetic inhibitor, nikkomycin Z, suppressed incorporation of GlcNAc by Pc preparations. Finally, treatment of rats with Pneumocystis pneumonia using nikkomycin Z significantly reduced organism burdens. Taken together, these data support an important role for GlcNAc generation in the cell surface of Pneumocystis organisms.

Section: Detailed Experimental Proceduresmentioning

confidence: 99%

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

Am J Respir Cell Mol Biol

Hebrink

Jenson

et al. 2017

Self Cite

“…Rtt109 is subject to complex regulation by histone chaperones both in vitro and in vivo. One such chaperone, Asf1, enhances the in vitro HAT activity of Rtt109 in both yeast and P. carinii (30,31,52,68) and is required for H3K56ac in vivo (35). The addition of recombinant ScAsf1 to dH3-H4 tetramers significantly enhanced PjRtt109-catalyzed HAT activity in vitro (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Garcinol is a polyisoprenylated benzophenone natural product that is reported to inhibit several HATs, including p300/CBP-associated factor (PCAF) and p300, in the low micromolar range in vitro (69,70). We recently showed that garcinol inhibits PcRtt109 HAT activity and yeast Rtt109-Vps75 in vitro at low micromolar concentrations (31,42). Of note, we observed that garcinol inhibited PjRtt109 HAT activity in vitro in a dose-dependent manner (IC 50 , 2.7 Ϯ 0.72 M) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Recombinant Schizosaccharomyces pombe Rtt109 (SpRtt109), PjRtt109, PcRtt109, REG␣, and glutathione S-transferase (GST) proteins were produced using standard procedures (30,31). Briefly, full-length cDNAs were amplified from cDNA using Pfu DNA polymerase.…”

Section: Methodsmentioning

confidence: 99%

“…In this light, we recently characterized the histone acetyltransferase (HAT) Rtt109 in P. carinii (30,31). Rtt109 HATs were first discovered in Saccharomyces cerevisiae because they catalyze a specific atypical posttranslational histone modification, i.e., histone H3 lysine 56 acetylation (H3K56ac) (32)(33)(34)(35).…”

mentioning

confidence: 99%

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

Dahlin

Antimicrob Agents Chemother

Han

et al. 2014

Self Cite

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.

Pathobiology ofPneumocystispneumonia: life cycle, cell wall and cell signal transduction

Skalski

Limper

2015

FEMS Yeast Research

Pneumocystis is a genus of ascomycetous fungi that are highly morbid pathogens in immunosuppressed humans and other mammals. Pneumocystis cannot easily be propagated in culture, which has greatly hindered understanding of its pathobiology. The Pneumocystis life cycle is intimately associated with its mammalian host lung environment, and life cycle progression is dependent on complex interactions with host alveolar epithelial cells and the extracellular matrix. The Pneumocystis cell wall is a varied and dynamic structure containing a dominant major surface glycoprotein, β-glucans and chitins that are important for evasion of host defenses and stimulation of the host immune system. Understanding of Pneumocystis cell signaling pathways is incomplete, but much has been deduced by comparison of the Pneumocystis genome with homologous genes and proteins in related fungi. In this mini-review, the pathobiology of Pneumocystis is reviewed, with particular focus on the life cycle, cell wall components and cell signal transduction.