Pneumocystis jirovecii Rtt109, a Novel Drug Target for Pneumocystis
Pneumonia in Immunosuppressed Humans
Jayme L. Dahlin,a,b Theodore Kottom,c,d Junhong Han,d Hui Zhou,d Michael A. Walters,e Zhiguo Zhang,d Andrew H. Limperc,d,f
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine, Rochester, Minnesota, USAa; Medical Scientist Training
Program, Mayo Clinic College of Medicine, Rochester, Minnesota, USAb; Thoracic Diseases Research Unit, Mayo Clinic College of Medicine, Rochester, Minnesota, USAc;
Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota, USAd; Institute for Therapeutics Discovery and Development,
University of Minnesota, Minneapolis, Minnesota, USAe; Division of Pulmonary and Critical Care, Department of Internal Medicine, Mayo Clinic College of Medicine,
Rochester, Minnesota, USAf
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.
P
neumocystis pneumonia (PcP) is a significant cause of morbidity
and mortality among patients with HIV infection or other immunosuppressive conditions (1–4). The incidence of PcP has risen significantly among certain non-HIV patients due to the increased use
of immunosuppressive therapies related to the management of organ
transplantation, autoimmune diseases, and cancer (5–7). The reported mortality rates for PcP range between 10 and 30% for AIDS
patients and between 30 and 70% for selected non-HIV-infected patients with immunosuppression (8–13). Several factors contribute to
poor PcP outcomes, including delayed diagnosis (14, 15) and complex host-pathogen interactions (16–21). Like other opportunistic
fungal pathogens, there is also the emerging threat of Pneumocystis
populations developing resistance to the currently available therapeutic agents (22–24). In humans, PcP is caused by the opportunistic fungal species Pneumocystis jirovecii, which specifically infects human hosts and is not viable in other immunosuppressed
mammalian hosts. Unfortunately, research progress has been hindered by the lack of continuous in vitro propagation methods for
Pneumocystis, which limits ready access to viable organisms for
laboratory research and drug discovery studies (25, 26). As a result, most drug discovery for PcP has been performed with other
species of Pneumocystis, such as Pneumocystis carinii or Pneumocystis murina generated in rats or mice, respectively (27–29).
However, questions remain regarding whether agents identified as
having anti-Pneumocystis activity against P. carinii possess critical
activity against the causal pathogen in human disease, namely, P.
jirovecii. Validation of anti-P. jirovecii drug activity and fundamental target characterization studies represent critical steps in
the process of therapeutic target validation.
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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) (32–35). Rtt109-catalyzed
H3K56ac occurs during the S phase of the cell cycle and promotes
genotoxic resistance, as it is associated with DNA replication and
DNA repair (34–37). Rtt109 homologues have been found widely
across the fungal kingdom, but no sequence homologies have
been found in humans, making these potentially attractive targets
for antifungal drug development. In humans and other mammals,
H3K56ac is catalyzed by the HATs p300/CREB-binding protein
(CBP) or GCN5 (38, 39). Additional evidence indicates that deletion of rtt109 in the opportunistic fungal pathogen Candida albicans reduces fungal infection burdens in mouse models (40, 41).
On this basis, we postulate that specific inhibitors of fungal
Rtt109-catalyzed histone acetylation may be useful as novel antifungal agents, with minimal mammalian toxicities, and may have
activity against recalcitrant organisms such as P. jirovecii (42,
43). Pneumocystis is challenging to treat with standard antifun-
Antimicrobial Agents and Chemotherapy
Received 24 February 2014 Returned for modification 12 March 2014
Accepted 11 April 2014
Published ahead of print 14 April 2014
Address correspondence to Andrew H. Limper, limper.andrew@mayo.edu.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.02637-14
p. 3650 –3659
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Pneumocystis jirovecii Rtt109
gals, and the use of the available agents can be limited by drugrelated toxicities. Accordingly, efficacious anti-Pneumocystis
agents with minimal human toxicities still represent an unmet
clinical need, despite significant efforts over the course of several
decades (44–49).
In the current investigation, we show that P. jirovecii expresses
a functional Rtt109 HAT (i.e., P. jirovecii Rtt109 [PjRtt109]). We
confirm the location of the Pjrtt109 gene within the recently sequenced P. jirovecii genome. Using heterologous expression, we
demonstrate that Pjrtt109 restores H3K56ac levels and genotoxic
resistance in rtt109-null yeast. PjRtt109 protein exhibits HAT activity in vitro, and this activity is enhanced by the addition of the
histone chaperone Asf1. Finally, we demonstrate that PjRtt109
enzymatic activity can be inhibited by reported small-molecule
HAT inhibitors, one of which reduces the viability of Pneumocystis
organisms. Both PjRtt109 and P. carinii Rtt109 (PcRtt109) were
inhibited by low micromolar concentrations of these two compounds in vitro. Together, these results demonstrate that PjRtt109
is a functional Rtt109 HAT, representing an attractive target for
therapeutic development targeting human PcP.
MATERIALS AND METHODS
Generation of full-length Pjrtt109 cDNA and subcloning into expression vectors. The full-length 1,143-bp Pjrtt109 cDNA was synthesized
commercially (GenScript USA) and subcloned into pUC57. This plasmid
containing the full-length Pjrtt109 cDNA reading frame was then used as
a template in PCRs using Pfu DNA polymerase (Life Technologies). The
cDNA was then cloned into the yeast pYES2.1 TOPO or pGEX-4T1 bacterial expression vector. Induction of gene expression in both bacteria and
yeast has been described previously (30).
Verification of Pjrtt109 in P. jirovecii genome. The PCR with the
partial Pjrtt109 DNA sequence was conducted using standard protocols.
Briefly, total genomic DNA from P. jirovecii was recovered from bronchoalveolar lavage (BAL) fluid specimens from potential positive cases of
Pneumocystis pneumonia. We obtained clinical waste BAL fluid samples
after all clinical diagnostic testing had been performed. We used the entire
residual samples (generally ⬍5 ml) to isolate P. jirovecii organisms, as
described previously (30). The entire P. jirovecii isolate was lysed in toto,
and nucleic acids were extracted. Freshly isolated P. jirovecii genomic
DNA was prepared with the IsoQuick nucleic acid extraction kit (Orca
Research). After isolation, approximately 250 ng of DNA was used in a
PCR utilizing Pfu DNA polymerase with the following Pjrtt109 gene-specific primers: forward, 5=-TGGTGGGCAAAAGTGTTGG-3=; reverse, 5=GTGTCTCAAAATCAGAACGC-3=. To verify that these primers would
not amplify segments of human genomic DNA, the primer set was also
tested against human DNA isolated from healthy lung cells (Amsbio).
Using another set of specific primers, the human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) gene was amplified to verify that this
DNA supply was not degraded. The sequences for the hGAPDH primers
were as follows: forward, 5=-CGGATTTGGTCGTATTGGGC-3=; reverse,
5=-TGGAAGATGGTGATGGGATTTC-3=.
Heterologous expression of Pjrtt109 in yeast. The BY4741 rtt109null (YLL002W, MATa his3⌬0 leu2⌬0 met15⌬0 ura3⌬0 rtt109⌬) S. cerevisiae strain was transformed with either a control vector (pYES2.1/V5-His/
lacZ) or pYES2.1 TOPO containing the in-frame full-length Pjrtt109
cDNA (pYES2.1/Pjrtt109). Expression of downstream Pjrtt109 cDNA was
under the control of the yeast GAL1 promoter, which can be induced with
the addition of 2% galactose to the medium. The parent strain BY4741
(MATa his3⌬0 leu2⌬0 met15⌬0 ura3⌬0) with pYES2.1/V5-His/lacZ was
used as the wild-type (WT) control. Cells were grown overnight at 30°C in
synthetic complete medium containing 2% glucose and supplemented
with appropriate amino acids but lacking uracil, to select and to maintain
the plasmids. S. cerevisiae strains were then grown overnight in liquid
minimal medium minus uracil and with 2% galactose in place of glucose.
July 2014 Volume 58 Number 7
Yeast whole-cell extracts were prepared using standard procedures. For
genotoxic sensitivity assays, yeast extracts were serially diluted to 1 ⫻ 106
cells/ml. We then plated 10 l of serial 10-fold dilutions of the indicated
yeast strains onto solid minimal medium lacking uracil and containing
2% galactose. Alternatively, yeast extracts were plated onto solid minimal
medium lacking uracil and containing 2% galactose plus one of the following DNA-damaging agents: 1 g/ml camptothecin (CPT), 50 mM
hydroxyurea (HU), or 0.005% methyl methanesulfonate (MMS). Cells
were grown for 72 h at 30°C and then assessed for growth by inspecting the
colony diameters.
Expression and purification of recombinant proteins. 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. Genes were further cloned into
the pGEX-4T1 vector, sequenced, and transformed into bacterial strain
BL21(DE3)pLys-S. GST-tagged proteins were produced overnight at
18°C by induction with 0.5 mM isopropyl -D-1-thiogalactopyranoside.
Culture broths (typically 1 to 2 liters) were centrifuged, and cell lysates
were obtained by passing the suspended pellets through a French press in
lysis buffer supplemented with protease inhibitors. The resulting lysates
were sonicated briefly and then centrifuged to remove insoluble debris.
The proteins were collected onto glutathione-Sepharose beads (GE
Healthcare), washed, and then eluted with a standard glutathione gradient. Eluted proteins were dialyzed overnight at 4°C in protein storage
buffer containing 10% glycerol (vol/vol) and 1 mM dithiothreitol (DTT).
SDS-PAGE, followed by Coomassie brilliant blue (CBB) staining, was
used to verify gross protein purity and the correct molecular weights of the
purified proteins. Drosophila histone tetramers (dH3–H4) were obtained
as previously described (50). Bovine serum albumin (BSA) (Sigma) was
used as a negative protein control in some experiments.
Histone acetyltransferase assays. HAT activity was measured in vitro
as previously reported, but with some minor modifications (34, 51). Reactions were performed in triplicate at 30°C for 30 min in 30-l volumes
containing final concentrations of 50 mM Tris-HCl (pH 8.0), 50 mM KCl,
0.1 mM EDTA, 1 mM DTT, 5 mM phenylmethylsulfonyl fluoride
(PMSF), 5 mM sodium butyrate, 0.01% Triton X-100 (vol/vol), and approximately 2.5 M [3H]acetyl-coenzyme A (PerkinElmer). Enzymes
were tested at approximately 800 nM concentrations, while recombinant
dH3–H4 tetramers (1.25 M) were used as the acetylation substrate. Reaction mixture aliquots (15 l) were immediately spotted onto Whatman
P-81 phosphocellulose paper filters (GE Healthcare) and air dried. Filter
papers were washed five times (5 min per cycle) with 50 mM NaHCO3
(pH 9.0), rinsed with acetone, and then allowed to air dry for 30 min.
[3H]Acetate incorporation was then measured with an LS6500 liquid
scintillation counter (Beckman-Coulter). Acetylated proteins were identified by autoradiography after the resolution of reaction mixture aliquots
on 15% SDS-PAGE gels. The gels were soaked in Amplify fluorographic
reagent (GE Healthcare) for 30 min and then dried under vacuum for 2 h
at 80°C. Films were then exposed to the gels at ⫺80°C, typically for 48 h.
The acetylation status of H3K56 was assessed by Western blotting of reaction samples as described above but using unlabeled acetyl-CoA (sodium salt, 10 M final concentration; Sigma) from stocks stored in 0.01 M
sodium acetate (pH 5.0). Membranes were imaged with a LI-COR Odyssey system, and data were analyzed using Image Studio software (LI-COR
Biosciences). Purified REG␣ was used as a negative enzymatic control
(30). Purified GST was included as an additional tag control.
Protein complex assays. Protein complexes were assembled using standard procedures. S. cerevisiae Asf1 (ScAsf1) was produced as described
previously (52). To obtain the ScAsf1-dH3–H4 complex, approximately
equimolar amounts of ScAsf1 and dH3–H4 (as determined by SDS-PAGE
separation and subsequent CBB staining) were incubated overnight at 4°C
and purified by gel filtration chromatography (52). Experiments were
performed as described above, except that enzymes were tested at approx-
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imately 400 nM and HAT reactions were allowed to proceed for up to
30 min.
Compounds and reagents. Garcinol, a natural product with reported
anti-HAT activity in vitro, was purchased as a solid powder (Enzo Life
Sciences) and was used without further purification. Compound 1 (PubChem compound identification 4785700), which has recently been reported to have selective activity against yeast Rtt109 in vitro, was also
obtained commercially as a solid powder (Enamine) and was used after
standard reverse-phase high-performance liquid chromatography (RPHPLC) purification (43). All solids showed greater than 98% purity in
ultra-performance liquid chromatography/mass spectrometry (UPLC/
MS) analyses, and their 1H and 13C NMR spectra were consistent with
their reported chemical structures. Compounds were prepared as 10 mM
stock solutions in dimethyl sulfoxide (DMSO) and were stored at ⫺20°C
under a vacuum seal. The 1H NMR (400 MHz) and 13C NMR (100 MHz)
spectra were recorded on a Bruker Avance spectrometer, while the
UPLC-MS analyses were performed using a Waters Acquity UPLC system
equipped with a ZQ mass spectrometer, a photodiode array, and evaporative light-scattering detectors.
Anti-HAT activity dose-response analyses. Garcinol and compound
1 were tested at up to 10 concentrations (final compound concentrations
of 20 nM to 250 M) using the aforementioned [3H]acetyl-CoA in vitro
HAT assay, with some minor modifications (42). Briefly, test compounds
were allowed to preequilibrate with the enzyme (approximately 400 nM)
and histone substrate for 5 min at 30°C before the HAT reaction was
initiated with the addition of [3H]acetyl-CoA. Reactions were performed
in 60-l total volumes. The DMSO content was kept constant at 3%
(vol/vol), and protease inhibitors were omitted from the reaction mixtures. The HAT reactions were allowed to proceed for 10 min, after which
reaction mixture aliquots were immediately spotted onto P-81 filter paper
and worked up as described above. Percent inhibition was calculated as a
percentage of the DMSO control value minus the background value.
Dose-response curves were generated in GraphPad Prism 6.0, using the
sigmoidal dose-response variable-slope four-parameter equation.
Effects of reported HAT inhibitors on Pneumocystis viability. Compound 1 and garcinol were incubated with freshly isolated P. carinii organisms maintained ex vivo in viability medium for 72 h. Relative viability was
assessed by measuring the ATP contents of the organisms using an ATP
bioluminescent assay, as described previously (53). The ATP assay measures the viability of mixed isolates of Pneumocystis trophic forms and
cysts. Approximately 5 ⫻ 107 organisms were tested under each condition, in RPMI 1640 medium containing 20% fetal bovine serum to promote viability. Each compound was tested in triplicate, and a total of three
independent experiments were performed using three separate isolations
of P. carinii organisms. As controls, P. carinii organisms were also maintained in medium alone, in medium containing 10 g/ml ampicillin
(Sigma), or in medium containing the amount of DMSO diluent required
to solubilize the test agents. In addition, pentamidine isethionate (Sigma)
was tested at 1 g/ml as a positive-control compound for anti-Pneumocystis activity. The organisms were incubated at 37°C with 5% CO2 in
standard 24-well plates. After 72 h, equal volumes (50 l) were removed
from each well, and the ATP levels were quantified using an ATPLite-M
kit (PerkinElmer).
Statistical analyses. All data are expressed as mean ⫾ standard deviation. Differences between groups were determined using one-way analysis of
variance (ANOVA) and multiple-comparison tests. Graphing and statistical
testing were performed using GraphPad Prism, with statistical differences
considered significant at P values of ⬍0.05, ⬍0.01, and ⬍0.001.
RESULTS
P. jirovecii expresses an Rtt109 gene orthologue. To address
whether P. jirovecii contains a potential Rtt109 HAT, we performed an in silico search of the recently reported P. jirovecii genome (54, 55). A putative Pjrtt109 orthologue (GenBank accession number CCJ28444) was identified in a pairwise alignment
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with PcRtt109 (GenBank accession number ACR39370.1), showing 61% primary sequence conservation (Fig. 1, top). The moderate divergence between these two closely related species is not unexpected, as several features in the mitochondrial DNA of P. murina and
P. carinii diverge from those of P. jirovecii (56), and this pattern of
sequence similarity has also been observed between P. carinii and
P. jirovecii dihydrofolate reductases (47, 57). In addition, there
appears to be significant genetic diversity at several regions among
P. jirovecii isolates, based on genetic analyses (58–61). The putative
PjRtt109 sequence showed several conserved regions in comparison
with other previously characterized fungal Rtt109 proteins, such as
those of Saccharomyces cerevisiae (GenBank accession number
Q07794), Schizosaccharomyces pombe (GenBank accession number Q9Y7Y5), and Candida albicans (GenBank accession number
Q5AAJ8) (Fig. 1, bottom). These regions included the aspartic
acid at amino acid position 84 in the conserved SKAD motif. Mutations of this specific residue result in nearly complete abolishment of in vitro H3 histone acetylation at the K56 position for both
PcRtt109 and S. cerevisiae Rtt109 (ScRtt109) (30, 34). As with
PcRtt109, the Pjrtt109 gene encodes a conserved lysine residue at
amino acid position 221, which is analogous to the site for autoacetylation in yeast Rtt109 (62, 63). Furthermore, P. murina
Rtt109 (GenBank accession number EMR10273) showed significant homology to both PjRtt109 and PcRtt109 (data not shown).
Despite difficulties associated with studying Pneumocystis organisms in the laboratory, additional evidence supports the idea that
Pjrtt109 is expressed by P. jirovecii. P. jirovecii organisms were
obtained from a BAL fluid sample from a patient with PcP, and
total RNA was extracted and amplified nonspecifically prior to
sequencing (54). A total of 56 RNA-sequencing reads mapped
unambiguously to the putative Pjrtt109 gene (http://www.ebi.ac
.uk/ena/data/view/ERP001479). Additionally, the Pjrtt109 gene
was present in the transcriptome assembly (HAAA01000299)
(http://www.ebi.ac.uk/ena/data/view/HAAA01000299), which was assembled de novo using RNA-sequencing reads (O. Cissé, personal communication).
With this information, we next sought to confirm the presence
of the putative Pjrtt109 gene in P. jirovecii organisms in vivo. To
accomplish this, we extracted P. jirovecii genomic DNA from BAL
fluid samples obtained from patients with confirmed cases of PcP
at the Mayo Medical Center. These Pneumocystis infections were
confirmed with our recently published single-copy nonnested
PCR assay that distinguishes active PcP from simple Pneumocystis
colonization (64). Primers based on the cDNA sequence of
Pjrtt109 were mixed with either P. jirovecii genomic DNA or human genomic DNA isolated from healthy human lung cells. As
expected, the Pjrtt109 primer set amplified a specific amplicon of
the expected size with P. jirovecii genomic DNA as the template
but not with human genomic DNA as the template (Fig. 2). As a
positive control for human DNA quality, we also included a
primer set based on the hGAPDH gene, which amplified an expected amplicon using human genomic DNA but not P. jirovecii
genomic DNA (Fig. 2). These results strongly support the idea that
P. jirovecii contains the Pjrtt109 gene and this gene is specifically
represented in the P. jirovecii genome.
Pjrtt109 restores H3K56ac levels and genotoxic resistance in
rtt109-null yeast. In S. cerevisiae, Rtt109 is required for H3K56ac
and is associated with genotoxic resistance due to its role in replication-coupled nucleosome assembly (34). Pneumocystis species
Antimicrobial Agents and Chemotherapy
Pneumocystis jirovecii Rtt109
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.
July 2014 Volume 58 Number 7
cannot be maintained using in vitro culture methods and cannot
yet be manipulated genetically. To circumvent these issues, our
group assesses Pneumocystis gene function using heterologous expression and complementation in other fungi such as S. cerevisiae
(65, 66). In this manner, we evaluated the potential activities of
Pjrtt109 in complementing the H3K56 acetylation defect in
rtt109-null yeast by transforming this strain with either pYES2.1/
V5-His/lacZ alone or the same vector containing full-length
Pjrtt109. Western blots of yeast whole-cell extracts demonstrated that
rtt109-null yeast cells were unable to acetylate H3K56. In contrast,
Pjrtt109 cDNA complementation efficiently restored H3K56ac levels in
rtt109-null yeast to levels seen in WT yeast (Fig. 3A).
In addition, rtt109-null yeast demonstrated increased sensitiv-
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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; ⌬, 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.
ity to genotoxins such as MMS, CPT, and HU (34, 35). As anticipated for a putative Rtt109 HAT, Pjrtt109 complementation restored genotoxin resistance in rtt109-null yeast to the level in WT
yeast (Fig. 3B). Taken together, these data strongly indicate that
Pneumocystis-derived Pjrtt109 can function in promoting
H3K56ac and genotoxin resistance in budding yeast, supporting
the notion that PjRtt109 may perform similar functions in P. jirovecii in vivo.
PjRtt109 is an active HAT in vitro. Based on the heterologous
expression experiments that demonstrated Pjrtt109 functionality
in vivo, we next sought to confirm the HAT activity of expressed
PjRtt109 protein in vitro. Purified PjRtt109 protein demonstrated
HAT activity comparable to that of its orthologue PcRtt109 in an
in vitro [3H]acetyl-CoA HAT assay that quantified the amount of
[3H]acetate incorporated into histone substrates (Fig. 4A). Autoradiographs of the reaction mixtures demonstrated that histone
acetylation was confined to histone H3 and not histone H4, consistent with the known substrate profiles of previously character-
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.
ⴱⴱⴱ, P ⬍ 0.001, compared with the REG␣ 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).
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Antimicrobial Agents and Chemotherapy
Pneumocystis jirovecii Rtt109
ized Rtt109 enzymes (Fig. 4B). Western blots verified that
PjRtt109 catalyzed H3K56ac in vitro (Fig. 4C). To show that
PjRtt109 was homologous to fungal Rtt109 HATs outside the
Pneumocystis genus, the HAT activities of Pneumocystis jirovecii
Rtt109 and Schizosaccharomyces pombe Rtt109 were also compared in vitro (30, 52, 67). The recombinant enzymes demonstrated comparable HAT activities (Fig. 4D). Autoradiographs
(Fig. 4E) and Western blots (Fig. 4F) showed that both Rtt109
proteins catalyzed acetylation on histone H3 and both proteins
were capable of acetylating H3K56 in vitro. Autoacetylation is an
important regulatory element in ScRtt109 (62, 63), and low-level
autoacetylation has been observed for PcRtt109 (30). As expected,
we observed a band consistent with PjRtt109 autoacetylation using extended-exposure autoradiographs, demonstrating that autoacetylation is likely a conserved feature of Rtt109 HATs (Fig.
4G). These results demonstrate that PjRtt109 possesses functional
Rtt109 HAT activity in vitro and that this activity is comparable to
that of other fungal Rtt109 HATs. Such information is vital for the
eventual development of Rtt109 inhibitors with the potential for
therapeutic activity across a variety of clinically relevant fungal
species that infect humans, such as P. jirovecii.
PjRtt109 HAT activity is enhanced by the histone chaperone
Asf1 in vitro. 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.
5A). As expected, autoradiography confirmed that the enhanced
acetylation was confined to histone H3 and not ScAsf1 or histone
H4 (Fig. 5B). Western blots confirmed that this ScAsf1-mediated
increase in PjRtt109-catalyzed histone acetylation in vitro led to an
increase in H3K56ac (Fig. 5C). These results demonstrate that,
like previously characterized Rtt109 HATs, PjRtt109 HAT activity
in vitro is enhanced by the histone chaperone Asf1.
PjRtt109 HAT activity is inhibited by small molecules in
vitro. Currently, there are no reports of small molecules that specifically inhibit PjRtt109 or PcRtt109 activity. In the absence of such
compounds, we examined the effects of several previously reported
HAT inhibitors on PjRtt109 enzymatic activity in vitro. 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 (IC50, 2.7 ⫾ 0.72 M) (Fig. 6A and
B). Compound 1 has been recently reported as a low-nanomolar
inhibitor of yeast Rtt109 in vitro and does not inhibit the HATs p300
and GCN5 in vitro (43). Like garcinol, compound 1 showed dosedependent inhibition of PjRtt109 HAT activity in vitro (IC50, 4.3 ⫾
3.9 M) (Fig. 6A and B). Fluconazole, an inhibitor of fungal cytochrome 14␣-demethylase with no reported activity against HATs,
was used as a negative-control compound. Both garcinol and compound 1 inhibited PjRtt109 and PcRtt109 at low micromolar concentrations (Fig. 6B). Using the reaction mixtures for garcinol, we confirmed dose-dependent decreases in histone acetylation via
autoradiography (Fig. 6C) and Western blotting for both H3K56ac
and H3K27ac (Fig. 6D). These results demonstrate that PjRtt109 is
capable of in vitro enzymatic inhibition by small molecules at low
July 2014 Volume 58 Number 7
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.
ⴱⴱⴱ, P ⬍ 0.001, pairwise comparisons between the dH3–H4 and ScAsf1dH3–H4 substrates for each enzyme or pairwise comparisons with the REG␣
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.
micromolar concentrations and that PjRtt109 and PcRtt109 are inhibited at similar compound concentrations under the conditions
tested.
Effects of small-molecule inhibitors of HAT activity on
Pneumocystis viability. Finally, as support of the concept, we
sought to investigate whether these reported small-molecule inhibitors of HAT activity might also alter Pneumocystis viability in vivo,
using a previously described ATP-based viability assay (53). To
investigate, we utilized freshly isolated P. carinii organisms, since
these are readily available in the laboratory and because our prior
experiments demonstrated comparable activities of the tested
HAT inhibitors against PcRtt109 and PjRtt109 in vitro. Interestingly, the general HAT inhibitor garcinol demonstrated dose-dependent suppression of P. carinii viability, as measured by ATP
aac.asm.org 3655
Dahlin et al.
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 g/ml)
and pentamidine (1 l/ml) were included as relevant negative- and positivecontrol 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. ⴱ, P ⬍ 0.05, compared with control ATP levels without test agent.
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 M) and compound 1 (IC50, 1.7 M). 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 PjRtt109catalyzed 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).
contents (Fig. 7). In contrast, the recently reported yeast Rtt109
inhibitor compound 1 failed to induce any reductions of P. carinii
viability under the conditions tested. Pentamidine and ampicillin
served as positive- and negative-control compounds, respectively.
The lack of observed activity for compound 1 is consistent with its
lack of activity against C. albicans in vivo (43). However, the activity of the reported HAT inhibitor garcinol to suppress Pneumocystis viability does support the further investigation of this class of
agents, as well as novel small molecules with more drug-like characteristics, potency, and target specificity, as potential antifungal
antimicrobials with activity against Pneumocystis species.
DISCUSSION
In the current study, we demonstrate that P. jirovecii contains a functional Rtt109 orthologue with functions parallel to those exhibited by
3656 aac.asm.org
PcRtt109, which is an important early characterization step in the
drug discovery process. We first identified the putative PjRtt109 orthologue by using a recently published P. jirovecii genome. We then
showed that a Pjrtt109-specific primer set specifically amplified a
product of the expected size when P. jirovecii genomic DNA was used
as the template but not when human genomic DNA was used, which
demonstrates that this gene is contained within the P. jirovecii genome. Because P. jirovecii cannot be cultured in vitro, we utilized
heterologous expression to show that Pjrtt109 restores genotoxin resistance and H3K56ac in rtt109-null yeast, which demonstrates its
functional importance as an Rtt109 HAT in this surrogate in vivo
system. We were further able to synthesize full-length Pjrtt109 cDNA
to study the function of recombinantly expressed PcRtt109 protein.
We verified that PjRtt109 has HAT activity in vitro, comparable to
that of its orthologue PcRtt109 and another fungal Rtt109, namely,
SpRtt109. As in previous reports describing PcRtt109, the HAT activity of PjRtt109 was enhanced by the addition of the yeast histone
chaperone Asf1, a salient feature of the well-characterized Rtt109Vps75-Asf1 system in yeast. Finally, since small-molecule inhibition
of Rtt109-catalyzed histone acetylation is hypothesized to be a potential novel epigenetically based antifungal therapy, we showed that the
reported HAT inhibitors garcinol and compound 1 can inhibit
PjRtt109 activity in vitro and these compounds inhibited PcRtt109
and PjRtt109 HAT activity comparably in vitro. Interestingly, garcinol, but not compound 1, also suppressed the viability of Pneumocystis organisms maintained ex vivo in viability medium.
Our observation that garcinol inhibits PjRtt109 and PcRtt109 as
well as other HATs raises interesting follow-up questions about the
target specificity of this natural product, its mechanism(s) of enzymatic inhibition, and its utility for additional in vivo studies. Garcinol
has been reported to inhibit Pneumocystis and yeast Rtt109, as well
as the HATs p300, PCAF, and GCN5, at low micromolar concentrations in vitro. It has also been reported to inhibit completely
unrelated enzymes in vitro (71). This behavior may be explained
by the chemical structure of garcinol, which contains an orthocatechol group that has the potential to oxidize to form a thiol-
Antimicrobial Agents and Chemotherapy
Pneumocystis jirovecii Rtt109
reactive ortho-quinone (72). Being aware of this possibility, we
have so far been unable to detect the presence of glutathionegarcinol adducts by UPLC-MS, although we admit that this does
not definitively exclude the possible formation of these adducts
(data not shown). Garcinol also can induce apoptosis, inhibit cancer cell growth, and modulate several important signaling pathways and has been shown to exert some protective effects (73–78).
With regard to potential mammalian toxicity, garcinol has been
studied in several animal experiments, in which it showed certain
chemoprotective effects and was relatively well-tolerated by rodents (79–81). The overall in vitro data are consistent with garcinol being a relatively nonselective inhibitor, and this property
may coincide with its ability to inhibit cancer cell growth in vitro.
Given its in vitro activity profile and the relatively high concentrations used in our fungal viability assays, we cannot exclude the
possibility of off-target effects contributing to the reduced viability of Pneumocystis upon exposure to garcinol. Therefore, we
think that garcinol is not ideal for further in vivo experiments in its
present form, because of its potential for off-target effects and the
need for more-definitive mechanistic studies. More specific and
potent chemical probes for HATs like Pneumocystis Rtt109 would
be useful to better assess our therapeutic hypothesis in a cellular
setting.
This demonstration that both PjRtt109 and PcRtt109 are functional HAT orthologues is significant for early Rtt109 drug discovery efforts for several reasons. First, it demonstrates that functional Rtt109 HATs are conserved across fungi. Second, it verifies
that, in the absence of P. jirovecii culture systems, P. carinii may
serve as an entirely suitable surrogate model for studying the
Rtt109 system in the laboratory setting. In addition, the observation that reported small-molecule HAT inhibitors can disrupt
PjRtt109 activity in vitro suggests that inhibitors developed for one
species of Rtt109 may inhibit homologues across a variety of other
fungal species, indicating that such a strategy may benefit a wide
range of fungal infections. The discovery and development of
more-potent small molecules targeting Rtt109 HATs will be useful
to more rigorously address this speculation. Finally, our findings
support further target characterization and drug development efforts with respect to PjRtt109, a potential epigenetic therapeutic
target with clinical relevance as the specific cause of human PcP.
This study strongly validates the use of the P. carinii system for
preclinical work of relevance for drug development eventually targeting human disease caused by P. jirovecii. We recently characterized the Rtt109-Vps75-Asf1 system in P. carinii, showing that
PcRtt109 restores genotoxic resistance and H3K56ac levels in
rtt109-null yeast. This system verifies many of the features found
in the well-characterized S. cerevisiae and S. pombe Rtt109-Vps75Asf1 systems in vitro, such as HAT activity being enhanced by the
histone chaperone Asf1. As Rtt109 is conserved in fungi but not in
mammals, specific inhibitors of Rtt109-catalyzed histone acetylation may eventually prove useful as novel antifungal targets across
a number of relevant infections, including P. jirovecii-related
pneumonia.
It is noteworthy that the general HAT inhibitor garcinol also
reduced the in vitro viability of Pneumocystis organisms. Interestingly, the recently reported specific Rtt109 inhibitor compound
1 did not exhibit any suppression of Pneumocystis viability. Similar
findings were noted when this compound was tested previously
against C. albicans (43). There are several possible reasons for our
observations, including possibly poor penetration of compound 1
July 2014 Volume 58 Number 7
into the fungal cytoplasm, the presence of active rapid export proteins that may limit internal accumulation of this agent, or compound instability under the testing conditions. Nonetheless, the
wide presence of Rtt109 proteins across pathogenic fungi, the divergence from mammalian host HAT proteins, and our initial
results on such agents reducing Pneumocystis viability continue to
support additional searches for more-selective anti-Rtt109 agents
that may be of therapeutic benefit for fungal infections in humans,
including P. jirovecii pneumonia.
ACKNOWLEDGMENTS
We acknowledge Jessica Strasser and Subhashree Francis for chemical
purification and characterization. We gratefully acknowledge Ousmane
Cissé and Philippe Hauser, Institute of Microbiology, University Hospital
Center (Lausanne, Switzerland), for assistance in verifying Pjrtt109 expression. The purified P. carinii organisms used in this study were generated with the assistance of Deanne Hebrink. Human-derived P. jirovecii
organisms were obtained from the Mayo Clinical Microbiology Laboratory, with the assistance of Nancy Wengenack. The samples were stored as
residual clinical waste specimens.
This work was supported by the Minnesota Partnership for Biotechnology and Medical Genomics (grant 73-01), the National Institutes of
Health (grant R01HL-62150 to A.H.L.), and the Mayo Foundation for
Medical Education and Research. J.L.D. was supported by the NIH Medical Scientist Training Program (grant T32 GM065841), a Pharmaceutical
Research and Manufacturers of America Foundation predoctoral pharmacology/toxicology fellowship, and the Mayo Foundation.
The funders had no role in study design, data collection or analysis, the
decision to publish, or preparation of the manuscript. The opinions and
assertions contained herein belong to the authors and are not necessarily
the official views of the funders.
We declare no conflicting interests.
J.L.D., T.K., A.H.L, and Z.Z. designed the experiments, J.L.D., T.K.,
J.H., and H.Z. performed the experiments, J.L.D., T.K., A.H.L., Z.Z., and
M.A.W. analyzed the data, A.H.L., Z.Z., and M.A.W. contributed equipment and reagents, J.L.D. wrote the manuscript, and J.L.D., T.K., A.H.L.,
Z.Z., and M.A.W. contributed with revisions.
REFERENCES
1. Huang L, Cattamanchi A, Davis JL, den Boon S, Kovacs J, Meshnick S,
Miller RF, Walzer PD, Worodria W, Masur H, International HIVAssociated Opportunistic Pneumonias (IHOP) Study, Lung HIV Study.
2011. HIV-associated Pneumocystis pneumonia. Proc. Am. Thorac. Soc.
8:294 –300. http://dx.doi.org/10.1513/pats.201009-062WR.
2. Carmona EM, Limper AH. 2011. Update on the diagnosis and treatment
of Pneumocystis pneumonia. Ther. Adv. Respir. Dis. 5:41–59. http://dx.doi
.org/10.1177/1753465810380102.
3. Krajicek BJ, Limper AH, Thomas CF. 2008. Advances in the biology, pathogenesis and identification of Pneumocystis pneumonia. Curr. Opin. Pulm. Med.
14:228 –234. http://dx.doi.org/10.1097/MCP.0b013e3282f94abc.
4. Cushion MT, Stringer JR. 2010. Stealth and opportunism: alternative lifestyles of
species in the fungal genus Pneumocystis. Annu. Rev. Microbiol. 64:431– 452.
http://dx.doi.org/10.1146/annurev.micro.112408.134335.
5. Tasaka S, Tokuda H. 2012. Pneumocystis jirovecii pneumonia in nonHIV-infected patients in the era of novel immunosuppressive therapies. J.
Infect. Chemother. 18:793– 806. http://dx.doi.org/10.1007/s10156-012
-0453-0.
6. Mekinian A, Durand-Joly I, Hatron P, Moranne O, Denis G, Queyrel V. 2011.
Pneumocystis jirovecii colonization in patients with systemic autoimmune
diseases: prevalence, risk factors of colonization and outcome. Rheumatology 50:569 –577. http://dx.doi.org/10.1093/rheumatology/keq314.
7. Schmoldt S, Schuhegger R, Wendler T, Huber I, Söllner H, Hogardt M,
Arbogast H, Heesemann J, Bader L, Sing A. 2008. Molecular evidence of
nosocomial Pneumocystis jirovecii transmission among 16 patients after
kidney transplantation. J. Clin. Microbiol. 46:966 –971. http://dx.doi.org
/10.1128/JCM.02016-07.
8. Ewig S. 1996. The effect of prophylaxis on the outcome of HIV-associated
aac.asm.org 3657
Dahlin et al.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
Pneumocystis carinii pneumonia. Chest 109:586 –587. http://dx.doi.org/10
.1378/chest.109.2.586.
Monnet X, Vidal-Petiot E, Osman D, Hamzaoui O, Durrbach A, Goujard C,
Miceli C, Bouree P, Richard C. 2008. Critical care management and outcome of
severe Pneumocystis pneumonia in patients with and without HIV infection. Crit. Care 12:R28. http://dx.doi.org/10.1186/cc6806.
Fei M, Kim E, Sant C, Jarlsberg L, Davis J, Swartzman A, Huang L.
2009. Predicting mortality from HIV-associated Pneumocystis pneumonia
at illness presentation: an observational cohort study. Thorax 64:1070 –
1076. http://dx.doi.org/10.1136/thx.2009.117846.
Fisk M, Sage E, Edwards S, Cartledge J, Miller R. 2009. Outcome from
treatment of Pneumocystis jirovecii pneumonia with co-trimoxazole. Int. J.
STD AIDS 20:652– 653. http://dx.doi.org/10.1258/ijsa.2009.008470.
Mansharamani N, Garland R, Delaney D, Koziel H. 2000. Management
and outcome patterns for adult Pneumocystis carinii pneumonia, 1985 to
1995: comparison of HIV-associated cases to other immunocompromised
states. Chest 118:704 –711. http://dx.doi.org/10.1378/chest.118.3.704.
Festic E, Gajic O, Limper A, Aksamit T. 2005. Acute respiratory failure
due to Pneumocystis pneumonia in patients without human immunodeficiency virus infection: outcome and associated features. Chest 128:573–
579. http://dx.doi.org/10.1378/chest.128.2.573.
Held J, Koch MS, Reischl U, Danner T, Serr A. 2011. Serum (1¡3)-D-glucan measurement as an early indicator of Pneumocystis jirovecii
pneumonia and evaluation of its prognostic value. Clin. Microbiol. Infect.
17:595– 602. http://dx.doi.org/10.1111/j.1469-0691.2010.03318.x.
Damiani C, Gal SL, Costa CD, Virmaux M, Nevez G, Totet A. 2013.
Combined quantification of pulmonary Pneumocystis jirovecii DNA and
serum (1¡3)--D-glucan for differential diagnosis of Pneumocystis pneumonia and Pneumocystis colonization. J. Clin. Microbiol. 51:3380 –3388.
http://dx.doi.org/10.1128/JCM.01554-13.
Steele C, Shellito JE, Kolls JK. 2005. Immunity against the opportunistic
fungal pathogen Pneumocystis. Med. Mycol. 43:1–19. http://dx.doi.org/10
.1080/13693780400015360.
Villegas LR, Kottom TJ, Limper AH. 2010. Characterization of PCEng2,
a -1,3-endoglucanase homolog in Pneumocystis carinii with activity in
cell wall regulation. Am. J. Respir. Cell Mol. Biol. 43:192–200. http://dx
.doi.org/10.1165/rcmb.2009-0131OC.
Gigliotti F, Wright T. 2005. Immunopathogenesis of Pneumocystis carinii
pneumonia. Expert Rev. Mol. Med. 7:1–16. http://dx.doi.org/10.1017
/S1462399405010203.
Kelly M, Shellito J. 2010. Current understanding of Pneumocystis immunology. Future Microbiol. 5:43– 65. http://dx.doi.org/10.2217/fmb.09.116.
Goodridge HS, Wolf AJ, Underhill DM. 2009. -Glucan recognition by
the innate immune system. Immunol. Rev. 230:38 –50. http://dx.doi.org
/10.1111/j.1600-065X.2009.00793.x.
Thomas CF, Jr, Limper AH. 2007. Current insights into the biology and
pathogenesis of Pneumocystis pneumonia. Nat. Rev. Microbiol. 5:298 –
308. http://dx.doi.org/10.1038/nrmicro1621.
Huang L, Crothers K, Atzori C, Benfield T, Miller R, Rabodonirina M,
Helweg-Larsen J. 2004. Dihydropteroate synthase gene mutations in
Pneumocystis and sulfa resistance. Emerg. Infect. Dis. 10:1721–1728. http:
//dx.doi.org/10.3201/eid1010.030994.
Kessl JJ, Hill P, Lange BB, Meshnick SR, Meunier B, Trumpower BL.
2004. Molecular basis for atovaquone resistance in Pneumocystis jirovecii
modeled in the cytochrome bc1 complex of Saccharomyces cerevisiae. J.
Biol. Chem. 279:2817–2824. http://dx.doi.org/10.1074/jbc.M309984200.
Walker DJ, Wakefield AE, Dohn MN, Miller RF, Baughman RP, Hossler PA, Bartlett MS, Smith JW, Kazanjian P, Meshnick SR. 1998.
Sequence polymorphisms in the Pneumocystis carinii cytochrome b gene
and their association with atovaquone prophylaxis failure. J. Infect. Dis.
178:1767–1775. http://dx.doi.org/10.1086/314509.
Sloand E, Laughon B, Armstrong M, Bartlett M, Blumenfeld W, Cushion M, Kalica A, Kovacs J, Martin W, Pitt E, Pesanti EL, Richards F,
Rose R, Walzer P. 1993. The challenge of Pneumocystis carinii culture. J.
Eukaryot. Microbiol. 40:188 –195. http://dx.doi.org/10.1111/j.1550-7408
.1993.tb04902.x.
Beck JM, Newbury RL, Palmer BE. 1996. Pneumocystis carinii pneumonia in scid mice induced by viable organisms propagated in vitro. Infect.
Immun. 64:4643– 4647.
Bartlett MS, Fishman JA, Durkin MM, Queener SF, Smith JW. 1990.
Pneumocystis carinii: improved models to study efficacy of drugs for treatment or prophylaxis of Pneumocystis pneumonia in the rat (Rattus
3658 aac.asm.org
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
spp.). Exp. Parasitol. 70:100 –106. http://dx.doi.org/10.1016/0014-4894
(90)90089-U.
McFadden DC, Powles MA, Pittarelli LA, Schmatz DM. 1991. Establishment of Pneumocystis carinii in various mouse strains using natural
transmission to initiate infection. J. Protozool. 38:126S–127S.
Beck JM, Preston AM, Wilcoxen SE, Morris SB, Sturrock A, Paine R.
2009. Critical roles of inflammation and apoptosis in improved survival in
a model of hyperoxia-induced acute lung injury in Pneumocystis murinainfected mice. Infect. Immun. 77:1053–1060. http://dx.doi.org/10.1128
/IAI.00967-08.
Kottom TJ, Han J, Zhang Z, Limper AH. 2011. Pneumocystis carinii
expresses an active Rtt109 histone acetyltransferase. Am. J. Respir. Cell
Mol. Biol. 44:768 –776. http://dx.doi.org/10.1165/rcmb.2009-0443OC.
Pupaibool J, Kottom TJ, Bouchonville K, Limper AH. 2013. Characterization of the Pneumocystis carinii histone acetyltranferase chaperone proteins PcAsf1 and PcVps75. Infect. Immun. 81:2268 –2275. http://dx.doi
.org/10.1128/IAI.01173-12.
Masumoto H, Hawke D, Kobayashi R, Verreault A. 2005. A role for
cell-cycle-regulated histone H3 lysine 56 acetylation in the DNA damage
response. Nature 436:294 –298. http://dx.doi.org/10.1038/nature03714.
Tsubota T, Berndsen CE, Erkmann JA, Smith CL, Yang L, Freitas MA,
Denu JM, Kaufman PD. 2007. Histone H3–K56 acetylation is catalyzed
by histone chaperone-dependent complexes. Mol. Cell 25:703–712. http:
//dx.doi.org/10.1016/j.molcel.2007.02.006.
Han J, Zhou H, Horazdovsky B, Zhang K, Xu R, Zhang Z. 2007. Rtt109
acetylates histone H3 lysine 56 and functions in DNA replication. Science
315:653– 655. http://dx.doi.org/10.1126/science.1133234.
Driscoll R, Hudson A, Jackson SP. 2007. Yeast Rtt109 promotes genome
stability by acetylating histone H3 on lysine 56. Science 315:649 – 652.
http://dx.doi.org/10.1126/science.1135862.
Chen CC, Carson JJ, Feser J, Tamburini B, Zabaronick S, Linger J, Tyler
JK. 2008. Acetylated lysine 56 on histone H3 drives chromatin assembly
after repair and signals for the completion of repair. Cell 134:231–243.
http://dx.doi.org/10.1016/j.cell.2008.06.035.
Li Q, Zhou H, Wurtele H, Davies B, Horazdovsky B, Verreault A,
Zhang Z. 2008. Acetylation of histone H3 lysine 56 regulates replicationcoupled nucleosome assembly. Cell 134:244 –255. http://dx.doi.org/10
.1016/j.cell.2008.06.018.
Das C, Lucia M, Hansen K, Tyler J. 2009. CBP/p300-mediated acetylation of histone H3 on lysine 56. Nature 459:113–117. http://dx.doi.org/10
.1038/nature07861.
Tjeertes J, Miller K, Jackson S. 2009. Screen for DNA-damage-responsive
histone modifications identifies H3K9Ac and H3K56Ac in human cells.
EMBO J. 28:1878 –1889. http://dx.doi.org/10.1038/emboj.2009.119.
Wurtele H, Tsao S, Lépine G, Mullick A, Tremblay J, Drogaris P, Lee
E-H, Thibault P, Verreault A, Raymond M. 2010. Modulation of histone
H3 lysine 56 acetylation as an antifungal therapeutic strategy. Nat. Med.
16:774 –780. http://dx.doi.org/10.1038/nm.2175.
Lopes da Rosa J, Boyartchuk VL, Zhu LJ, Kaufman PD. 2010. Histone
acetyltransferase Rtt109 is required for Candida albicans pathogenesis.
Proc. Natl. Acad. Sci. U. S. A. 107:1594 –1599. http://dx.doi.org/10.1073
/pnas.0912427107.
Dahlin JL, Sinville R, Solberg J, Zhou H, Han J, Francis S, Strasser JM, John K,
Hook DJ, Walters MA, Zhang Z. 2013. A cell-free fluorometric high-throughput
screen for inhibitors of Rtt109-catalyzed histone acetylation. PLoS One 8:e78877.
http://dx.doi.org/10.1371/journal.pone.0078877.
Lopes da Rosa J, Bajaj V, Spoonamore J, Kaufman PD. 2013. A small
molecule inhibitor of fungal histone acetyltransferase Rtt109. Bioorg.
Med. Chem. Lett. 23:2853–2859. http://dx.doi.org/10.1016/j.bmcl.2013
.03.112.
Gangjee A, Guo X, Queener SF, Cody V, Galitsky N, Luft JR, Pangborn
W. 1998. Selective Pneumocystis carinii dihydrofolate reductase inhibitors:
design, synthesis, and biological evaluation of new 2,4-diamino-5substituted-furo[2,3-d]pyrimidines. J. Med. Chem. 41:1263–1271. http:
//dx.doi.org/10.1021/jm970537w.
Anderson AC, Perry KM, Freymann DM, Stroud RM. 2000. The crystal
structure of thymidylate synthase from Pneumocystis carinii reveals a fungal insert important for drug design. J. Mol. Biol. 297:645– 657. http://dx
.doi.org/10.1006/jmbi.2000.3544.
Cushion MT, Walzer PD, Ashbaugh A, Rebholz S, Brubaker R, Eynde
JJV, Mayence A, Huang TL. 2006. In vitro selection and in vivo efficacy of
piperazine- and alkanediamide-linked bisbenzamidines against Pneumo-
Antimicrobial Agents and Chemotherapy
Pneumocystis jirovecii Rtt109
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
cystis pneumonia in mice. Antimicrob. Agents Chemother. 50:2337–2343.
http://dx.doi.org/10.1128/AAC.00126-06.
Cody V, Pace J, Queener SF, Adair OO, Gangjee A. 2013. Kinetic and
structural analysis for potent antifolate inhibition of Pneumocystis jirovecii, Pneumocystis carinii, and human dihydrofolate reductases and their
active-site variants. Antimicrob. Agents Chemother. 57:2669 –2677. http:
//dx.doi.org/10.1128/AAC.00172-13.
Porollo A, Meller J, Joshi Y, Jaiswal V, Smulian AG, Cushion MT. 2012.
Analysis of current antifungal agents and their targets within the Pneumocystis carinii genome. Curr. Drug Targets 13:1575–1585. http://dx.doi.org
/10.2174/138945012803530107.
Perez-Leal O, Moncada C, Clarkson AB, Merali S. 2011. Pneumocystis Sadenosylmethionine transport: a potential drug target. Am. J. Respir. Cell
Mol. Biol. 45:1142–1146. http://dx.doi.org/10.1165/rcmb.2011-0009OC.
Levenstein M, Kadonaga J. 2002. Biochemical analysis of chromatin
containing recombinant Drosophila core histones. J. Biol. Chem. 277:
8749 – 8754. http://dx.doi.org/10.1074/jbc.M111212200.
Berndsen CE, Denu JM. 2005. Assays for mechanistic investigations of
protein/histone acetyltransferases. Methods 36:321–331. http://dx.doi
.org/10.1016/j.ymeth.2005.03.002.
Han J, Zhou H, Li Z, Xu R, Zhang Z. 2007. Acetylation of lysine 56 of
histone H3 catalyzed by Rtt109 and regulated by Asf1 is required for replisome integrity. J. Biol. Chem. 282:28587–28596. http://dx.doi.org/10
.1074/jbc.M702496200.
Cushion MT, Collins M, Hazra B, Kaneshiro ES. 2000. Effects of atovaquone and diospyrin-based drugs on the cellular ATP of Pneumocystis
carinii f. sp. carinii. Antimicrob. Agents Chemother. 44:713–719. http://dx
.doi.org/10.1128/AAC.44.3.713-719.2000.
Cissé OH, Pagni M, Hauser PM. 2012. De novo assembly of the Pneumocystis jirovecii genome from a single bronchoalveolar lavage fluid specimen from a patient. mBio 4:e00428 –12. http://dx.doi.org/10.1128/mBio
.00428-12.
Cushion MT, Keely SP. 2013. Assembly and annotation of Pneumocystis
jirovecii from the human lung microbiome. mBio 4:e00224-13. http://dx
.doi.org/10.1128/mBio.00224-13.
Ma L, Huang D-W, Cuomo CA, Sykes S, Fantoni G, Das B, Sherman
BT, Yang J, Huber C, Xia Y, Davey E, Kutty G, Bishop L, Sassi M,
Lempicki RA, Kovacs JA. 2013. Sequencing and characterization of the
complete mitochondrial genomes of three Pneumocystis species provide
new insights into divergence between human and rodent Pneumocystis.
FASEB J. 27:1962–1972. http://dx.doi.org/10.1096/fj.12-224444.
Cody V, Pace J, Makin J, Piraino J, Queener SF, Rosowsky A. 2009.
Correlations of inhibitor kinetics for Pneumocystis jirovecii and human
dihydrofolate reductase with structural data for human active site mutant
enzyme complexes. Biochemistry 48:1702–1711. http://dx.doi.org/10
.1021/bi801960h.
Curran T, McCaughey C, Coyle PV. 2013. Pneumocystis jirovecii multilocus genotyping profiles in Northern Ireland. J. Med. Microbiol. 62:
1170 –1174. http://dx.doi.org/10.1099/jmm.0.057794-0.
Totet A, Duwat H, Magois E, Jounieaux V, Roux P, Raccurt C, Nevez
G. 2004. Similar genotypes of Pneumocystis jirovecii in different forms of
Pneumocystis infection. Microbiology 150:1173–1178. http://dx.doi.org
/10.1099/mic.0.26919-0.
Matos O, Esteves F. 2010. Pneumocystis jirovecii multilocus gene sequencing: findings and implications. Future Microbiol. 5:1257–1267. http://dx
.doi.org/10.2217/fmb.10.75.
Ripamonti C, Orenstein A, Kutty G, Huang L, Schuhegger R, Sing A,
Fantoni G, Atzori C, Vinton C, Huber C, Conville PS, Kovacs JA. 2009.
Restriction fragment length polymorphism typing demonstrates substantial diversity among Pneumocystis jirovecii isolates. J. Infect. Dis. 200:
1616 –1622. http://dx.doi.org/10.1086/644643.
Albaugh BN, Arnold KM, Lee S, Denu JM. 2011. Autoacetylation of the
histone acetyltransferase Rtt109. J. Biol. Chem. 286:24694 –24701. http:
//dx.doi.org/10.1074/jbc.M111.251579.
Stavropoulos P, Nagy V, Blobel G, Hoelz A. 2008. Molecular basis for the
autoregulation of the protein acetyl transferase Rtt109. Proc. Natl. Acad. Sci.
U. S. A. 105:12236–12241. http://dx.doi.org/10.1073/pnas.0805813105.
Wilson JW, Limper AH, Grys TE, Karre T, Wengenack NL, Binnicker MJ.
2011. Pneumocystis jirovecii testing by real-time polymerase chain reaction
and direct examination among immunocompetent and immunosuppressed patient groups and correlation to disease specificity. Diagn. Microbiol. Infect. Dis. 69:145–152. http://dx.doi.org/10.1016/j.diagmicrobio
.2010.10.021.
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65. Hauser P, Lo Presti L, Cockell M, Cerutti L, Simanis V. 2006. Analysis
of Pneumocystis carinii gene function by complementation in yeast mutants. J. Eukaryot. Microbiol. 53(Suppl 1):S149 –S150. http://dx.doi.org
/10.1111/j.1550-7408.2006.00210.x.
66. Grigore D, Meade JC. 2006. Functional complementation of the yeast
P-type H-ATPase, PMA1, by the Pneumocystis carinii P-type H-ATPase,
PCA1. J. Eukaryot. Microbiol. 53:157–164. http://dx.doi.org/10.1111/j
.1550-7408.2006.00089.x.
67. Xhemalce B, Miller KM, Driscoll R, Masumoto H, Jackson SP, Kouzarides T, Verreault A, Arcangioli B. 2007. Regulation of histone H3 lysine
56 acetylation in Schizosaccharomyces pombe. J. Biol. Chem. 282:15040 –
15047. http://dx.doi.org/10.1074/jbc.M701197200.
68. Adkins MW, Carson JJ, English CM, Ramey CJ, Tyler JK. 2007. The
histone chaperone anti-silencing function 1 stimulates the acetylation of
newly synthesized histone H3 in S-phase. J. Biol. Chem. 282:1334 –1340.
http://dx.doi.org/10.1074/jbc.M608025200.
69. Balasubramanyam K, Altaf M, Varier R, Swaminathan V, Ravindran A,
Sadhale P, Kundu T. 2004. Polyisoprenylated benzophenone, garcinol, a
natural histone acetyltransferase inhibitor, represses chromatin transcription and alters global gene expression. J. Biol. Chem. 279:33716 –33726.
http://dx.doi.org/10.1074/jbc.M402839200.
70. Arif M, Pradhan SK, Thanuja GR, Vedamurthy BM, Agrawal S, Dasgupta D, Kundu TK. 2009. Mechanism of p300 specific histone acetyltransferase inhibition by small molecules. J. Med. Chem. 52:267–277.
http://dx.doi.org/10.1021/jm800657z.
71. Koeberle A, Northoff H, Werz O. 2009. Identification of 5-lipoxygenase
and microsomal prostaglandin E2 synthase-1 as functional targets of the
anti-inflammatory and anti-carcinogenic garcinol. Biochem. Pharmacol.
77:1513–1521. http://dx.doi.org/10.1016/j.bcp.2009.02.005.
72. Baell JB, Holloway GA. 2010. New substructure filters for removal of pan
assay interference compounds (PAINS) from screening libraries and for
their exclusion in bioassays. J. Med. Chem. 53:2719 –2740. http://dx.doi
.org/10.1021/jm901137j.
73. Ahmad A, Sarkar SH, Bitar B, Ali S, Aboukameel A, Sethi S, Li Y, Bao
B, Kong D, Banerjee S, Padhye SB, Sarkar FH. 2012. Garcinol regulates
EMT and Wnt signaling pathways in vitro and in vivo, leading to anticancer activity against breast cancer cells. Mol. Cancer Ther. 11:2193–2201.
http://dx.doi.org/10.1158/1535-7163.MCT-12-0232-T.
74. Weng M-S, Liao C-H, Yu S-Y, Lin J-K. 2011. Garcinol promotes neurogenesis in rat cortical progenitor cells through the duration of extracellular signal-regulated kinase signaling. J. Agric. Food Chem. 59:1031–
1040. http://dx.doi.org/10.1021/jf104263s.
75. Cheng A-C, Tsai M-L, Liu C-M, Lee M-F, Nagabhushanam K, Ho C-T,
Pan M-H. 2010. Garcinol inhibits cell growth in hepatocellular carcinoma
Hep3B cells through induction of ROS-dependent apoptosis. Food Funct.
1:301–307. http://dx.doi.org/10.1039/c0fo00134a.
76. Hong J, Kwon SJ, Sang S, Ju J, Zhou JN, Ho CT, Huang MT, Yang CS.
2007. Effects of garcinol and its derivatives on intestinal cell growth: inhibitory
effects and autoxidation-dependent growth-stimulatory effects. Free Radic.
Biol. Med. 42:1211–1221. http://dx.doi.org/10.1016/j.freeradbiomed.2007.01
.016.
77. Liao CH, Sang S, Ho CT, Lin JK. 2005. Garcinol modulates tyrosine
phosphorylation of FAK and subsequently induces apoptosis through
down-regulation of Src, ERK, and Akt survival signaling in human colon
cancer cells. J. Cell. Biochem. 96:155–169. http://dx.doi.org/10.1002/jcb
.20540.
78. Oike T, Ogiwara H, Torikai K, Nakano T, Yokota J, Kohno T. 2012.
Garcinol, a histone acetyltransferase inhibitor, radiosensitizes cancer cells
by inhibiting non-homologous end joining. Int. J. Radiat. Oncol. Biol.
Phys. 84:815– 821. http://dx.doi.org/10.1016/j.ijrobp.2012.01.017.
79. Chen X, Zhang X, Lu Y, Shim J-Y, Sang S, Sun Z, Chen X. 2012.
Chemoprevention of 7,12-dimethylbenz[a]anthracene (DMBA)-induced
hamster cheek pouch carcinogenesis by a 5-lipoxygenase inhibitor, garcinol. Nutr. Cancer 64:1211–1218. http://dx.doi.org/10.1080/01635581
.2012.718032.
80. Yoshida K, Tanaka T, Hirose Y, Yamaguchi F, Kohno H, Toida M, Hara
A, Sugie S, Shibata T, Mori H. 2005. Dietary garcinol inhibits 4-nitroquinoline 1-oxide-induced tongue carcinogenesis in rats. Cancer Lett.
221:29 –39. http://dx.doi.org/10.1016/j.canlet.2004.08.016.
81. Tanaka T, Kohno H, Shimada R, Kagami S, Yamaguchi F, Kataoka S, Ariga
T, Murakami A, Koshimizu K, Ohigashi H. 2000. Prevention of colonic
aberrant crypt foci by dietary feeding of garcinol in male F344 rats. Carcinogenesis 21:1183–1189. http://dx.doi.org/10.1093/carcin/21.6.1183.
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