Development of a Ligand-Directed Approach To Study the Pathogenesis of Invasive Aspergillosis

Michail S Lionakis; Johanna Lahdenranta; Jessica Sun; Wei Liu; Russell E Lewis; Nathaniel D Albert; Renata Pasqualini; Wadih Arap; Dimitrios P Kontoyiannis

doi:10.1128/IAI.73.11.7747-7758.2005

. 2005 Nov;73(11):7747–7758. doi: 10.1128/IAI.73.11.7747-7758.2005

Development of a Ligand-Directed Approach To Study the Pathogenesis of Invasive Aspergillosis

Michail S Lionakis ¹, Johanna Lahdenranta ^2,3, Jessica Sun ^2,3, Wei Liu ¹, Russell E Lewis ^1,4, Nathaniel D Albert ¹, Renata Pasqualini ^2,3, Wadih Arap ^2,3,^*, Dimitrios P Kontoyiannis ^1,4,^*

PMCID: PMC1273901 PMID: 16239579

Abstract

Invasive aspergillosis is a leading cause of infectious death in immunosuppressed patients. Here, we adapted a phage display library-based selection to screen and identify binding peptides to the surface of Aspergillus fumigatus conidia and hyphae. We identified a peptide (sequence CGGRLGPFC) that reliably binds to the surface of Aspergillus fumigatus hyphae. Binding was not Aspergillus strain specific, as it was also observed in hyphae of other Aspergillus clinical isolates. Furthermore, CGGRLGPFC-displaying phage targets Aspergillus fumigatus hyphae on formalin-fixed paraffin-embedded histopathology sections of lung tissue recovered from mice with invasive pulmonary aspergillosis. This approach may yield reagents such as peptidomimetics for novel diagnostic and therapeutic interventions in invasive aspergillosis.

Invasive aspergillosis, mainly caused by Aspergillus fumigatus, is the most common opportunistic mycosis in immunosuppressed patients with leukemia, bone marrow and solid-organ transplant recipients and a frequent cause of morbidity and mortality (17, 20). As the pathogenesis of invasive aspergillosis involves inhalation of airborne conidia in susceptible hosts, pneumonia is the most common clinical manifestation (17, 20). As in other filamentous molds, there are two developmental programs of growth of Aspergillus species: conidia and hyphae (4, 20). Aspergillus conidia are continuously inhaled from the environment and, in normal hosts, most are efficiently phagocytosed by resident lung macrophages (4, 20). Some conidia escape phagocytosis and germinate to hyphae, the invasive form of Aspergillus species. Hyphae are then destroyed by neutrophils in immunocompetent hosts (4, 20).

The high mortality of invasive aspergillosis reflects the severe net state of immunosuppression of affected patients, delayed diagnosis, and the suboptimal in vivo efficacy of antifungal agents against Aspergillus species (17, 20). Hence, the combination of early detection along with targeted delivery of antifungal agents to the site of infection early on, when the tissue fungal burden is relatively low, could be of critical importance in improving the outcome of invasive aspergillosis.

Biopanning and Rapid Analysis of Selective Interactive Ligands (BRASIL) is an approach for the screening and identification of cell-surface-binding peptides from phage display libraries (10). Phage display technology has been used to identify vascular receptors in tumors and to characterize protein interacting sites in the context of mammalian cells (2, 13, 14, 27). However, this methodology has not as yet been applied to the study of infections such as invasive aspergillosis. We hypothesized that the surfaces of conidia and hyphae of Aspergillus fumigatus may contain binding sites that can be accessible and targeted by binding peptides.

In the present study, we first identify peptide ligands that bind in vitro to the surface of conidia and hyphae of Aspergillus fumigatus. Then, we show that a cyclic peptide motif (sequence CGGRLGPFC) binds to hyphae of several clinical mold isolates in vitro and to Aspergillus fumigatus hyphal elements present in lung tissue recovered from a murine model of invasive pulmonary aspergillosis. These data suggest that a ligand-directed system to targeting lesions in invasive aspergillosis can be developed by a combinatorial selection approach.

MATERIALS AND METHODS

Fungal isolates.

We tested the Aspergillus fumigatus clinical isolate 293 (AF293) used in the Aspergillus sequencing project (http://www.sanger.ac.uk/Projects/A_fumigatus/) (7). We also tested a panel of mold isolates (three Aspergillus fumigatus isolates [AF66, AF76, and AF101], two Aspergillus terreus isolates [AT11 and AT48], two Aspergillus flavus isolates [AF117 and AF122], two Aspergillus niger isolates [AN42 and AN75], and two Rhizopus isolates [Z160 and Z161]) recovered from cancer patients presenting with invasive mycoses at The University of Texas M. D. Anderson Cancer Center.

To obtain conidia, AF293 was plated on yeast agar glucose plates (0.5% yeast extract, 1.0% dextrose, 0.2% vitamin mixture, 0.1% trace elements, 1.5% agar, 1% MgSO₄) at 37°C; conidia were harvested 3 days later, counted by using hemocytometry, and suspended in phosphate-buffered saline (PBS). To obtain hyphae, AF293 conidia (suspensions of 10⁴ conidia/ml) were incubated in liquid yeast agar glucose medium at 37°C for 16 to 20 h, which allowed the transformation of >95% of conidia to hyphae with lengths varying from 50 to 70 μm as determined by using an inverted microscope. Following centrifugation, AF293 hyphae were suspended in PBS.

Biopanning and rapid analysis of selective interactive ligands (BRASIL).

We screened a phage display library for AF293 conidium and hypha binding ligands by the BRASIL method (1, 10). The phage library that was used in the present study contained the insert CX₇C (C, cysteine; X, any amino acid residue). It was derived from a large-scale preparation of a phage random peptide library and was designed to display a constrained cyclic loop within the pIII capsid protein (28, 33). The preparation was optimized to create the highest possible insert diversity; thus, the diversity of displayed peptides in the library was approximately 10⁸ to 10⁹ (1). All phage clones of the library are evaluated in vitro to rule out a selective growth advantage (rather than binding capability) that may explain the enrichment observed during the rounds of panning. This is obviously a routine control in all experiments with phage display screenings. As such, there is no growth advantage for any of the clones in this library.

Conidia suspended in PBS (10⁹ conidia/ml) or hyphae (a 100-μl aliquot from the concentrated hyphal PBS solution derived from centrifugation) were incubated with 10⁹ transducing units (TU) of the phage library (10). The conidium- or hypha-phage coincubation was performed in the presence of PBS containing 1% bovine serum albumin on ice to prevent or minimize nonspecific internalization. Following a 16-h coincubation, the suspension was transferred to the top of a nonmiscible organic lower phase with an intermediate specific density and centrifuged (10). In pilot experiments, we first determined the optimal phage separation conditions with a dibutyl phtalate:cyclohexane (Sigma Chemical Co., St. Louis, MO) ratio of 6:1. Also, hyphae required greater centrifugation forces (100,000 × g versus 10,000 × g for conidia) for longer times (20 min versus 10 min for conidia). Following centrifugation, conidia or hyphae entered the lower organic phase and pelleted at the bottom of the tube, carrying with them only specifically bound phage. The pellet was then transferred to another clean tube as previously described (10).

Phage bound to conidia or hyphae were rescued by infection of log-phase Escherichia coli K91kan bacteria (10). Briefly, following coincubation of Escherichia coli K91kan with conidium- or hypha-bound phage for 30 min, the infected bacteria were initially incubated in Luria-Bertani (LB) liquid medium containing kanamycin for selection of Escherichia coli K91kan bacteria, tetracycline (low concentration, 0.2 μg/ml) for the genes responsible for tetracycline resistance in the host bacteria to be induced (phage contains the tetracycline resistance gene) (1, 29, 33), and voriconazole (4 μg/ml). Voriconazole, an antifungal agent with good in vitro and in vivo activity against Aspergillus species (26), was used to suppress subsequent conidial and hyphal growth. Following that, bacteria were incubated overnight at 37°C in LB liquid medium containing kanamycin, tetracycline (high concentration; 40 μg/ml, for the selection of the phage-infected Escherichia coli K91kan bacteria), and voriconazole. The next day, amplified phage was separated from Escherichia coli K91kan bacteria by polyethylene glycol-NaCl precipitation and used in subsequent selection rounds (titer, 10⁹ TU) to enrich for the phage binding specifically to conidia or hyphae (10). After four rounds of selection for AF293 conidia and five rounds of selection for AF293 hyphae, we sequenced the DNA corresponding to peptide inserts of randomly picked phage clones for AF293 conidia and hyphae. Peptide sequences were then analyzed according to enrichment and by Clustal W sequence alignment. Selected motifs were used to search nonredundant protein databanks (National Center for Biotechnology Information [NCBI] BLAST [http://www.ncbi.nlm.nih.gov/BLAST]).

To evaluate phage binding, we used the fd-tet insertless phage as a negative control (1, 10). In parallel experiments, we coincubated conidia or hyphae with selected peptide-displaying or control phage and quantified phage bound to conidia or hyphae relative to fd-tet. We tested (in triplicate) various experimental conditions in independent experiments (i.e., phage inputs of 10⁶ to 10¹⁰ TU, dibutyl phtalate: cyclohexane ratios of 6:1 versus 9:1, and incubation periods of 4 h versus 16 h).

Murine model of invasive pulmonary aspergillosis.

The binding of CGGRLGPFC-displaying phage (relative to fd-tet binding) on AF293 hyphae was assessed on formalin-fixed, paraffin-embedded histopathology sections of lung tissue from mice with invasive pulmonary aspergillosis. This specific peptide-displaying phage was used because it was the one enriched in the screening and for which homology searches suggested that it corresponded to molecules of the extracellular matrix present in the lung parenchyma. Female Swiss Webster mice weighting 20 to 25 g each (Harlan Sprague-Dawley, Inc., Indianapolis, IN) were immunosuppressed with intraperitoneal cyclophosphamide (Sigma) (150 mg/kg of body weight; 200 to 250 μl of a 15-mg/ml sterile saline solution) and infected intranasally with AF293 conidia (inoculum, 35 μl of a 10⁹ conidia/ml solution or ∼35 × 10⁶ conidia per mouse) (21). A hyperacute pneumonia ensues, and mice typically succumb to their infection within 4 days (21). We euthanized the mice 4 days postinfection by CO₂ narcosis and cervical dislocation, removed their lungs, fixed the lungs with 10% formaldehyde, and embedded them in paraffin wax.

Phage overlay assays and immunohistochemistry studies.

Lung tissue sections (each, 5 μm) were deparaffinized in xylene and rehydrated in a graded alcohol series. Endogenous peroxidase was blocked by PBS containing 3% hydrogen peroxide for 10 min at room temperature (RT). For epitope retrieval, tissue sections were heated in target retrieval solution (DakoCytomation, Carpinteria, CA) in a standard steamer for 30 min and then allowed to cool to RT for 15 min. After equilibration with Tris-buffered saline containing 0.05% Tween 20, the slides were incubated with protein-blocking solution (DakoCytomation) for 15 min to block nonspecific protein binding sites (32). Subsequently, 2 × 10⁹ TU of the CGGRLGPFC-displaying or control phage were added and incubated for 2 h at RT. An anti-phage antibody (Sigma) (10 μg/ml; 200-μl volume of a 1:500 dilution in antibody diluent; DakoCytomation), or rabbit immunoglobulin G (IgG, negative control; DakoCytomation) was then added to the slides and incubated overnight at 4°C. After three washes with Tris-buffered saline containing 0.05% Tween 20, the peroxidase-conjugated anti-rabbit secondary antibody was added for 20 min at RT. Detection of immunoreaction was then achieved by using the LSAB+ system (DakoCytomation) containing streptavidin-biotin-peroxidase complex for 20 min incubation at RT (32). Color was developed with 3,3′-diaminobenzidine and hydrogen peroxide (DakoCytomation), and slides were subsequently counterstained with 100% hematoxylin, dehydrated, and mounted (32). At least two different sections from each mouse with macroscopic hemorrhagic lesions consistent with invasive pulmonary aspergillosis were tested per experiment. Each experiment was repeated independently two times on different days by similar methods.

Immunofluorescence studies.

Steps until the addition of anti-phage antibody (or rabbit IgG negative control) were performed as described above for immunohistochemistry studies. After an overnight incubation at 4°C and three washes with PBS, the tissue sections were incubated with fluorescence-labeled secondary donkey anti-rabbit antibody (Molecular Probes, Eugene, OR) at a concentration of 1:200 for 1 h and then counterstained with DAPI (4′,6′-diamidino-2-phenylindole; Molecular Probes) for 1 min (19). Subsequently, tissue sections were washed with PBS and mounted with Vectorshield mounting medium (Vector Laboratories, Burlingame, CA) (19). In control experiments, we tested for potential cross-reactivity of anti-phage or the secondary anti-rabbit antibody with Aspergillus hyphae. At least two different histopathology sections from each mouse with invasive pulmonary aspergillosis were tested per experiment. Each experiment was repeated two times independently on different days.

Statistical analysis.

Differences in the number of bound phages recovered from various binding experiments were analyzed by using the Mann-Whitney two-tailed t test (GraphPad Prism 3 software program; GraphPad Software, Inc., San Diego, California). P values of ≤0.05 were considered statistically significant.

RESULTS

Selection of peptide motifs that bind to the surface of Aspergillus fumigatus conidia and hyphae. Analysis of the peptides recovered from phage selection for Aspergillus conidia or hyphae according to enrichment and by Clustal W sequence alignment revealed that several amino acid residues were shared among multiple motifs (Table 1). Searches for each of these peptides in NCBI BLAST revealed that some corresponded or matched to motifs contained in human proteins (Tables 2 and 3).

TABLE 1.

Analysis of the peptides recovered from phage selection for Aspergillus conidia or hyphae according to enrichment and by Clustal W sequence alignment^a

Motif	Panning	Alignment	Motif	Panning	Alignment
G[HR][RS]R[DL]E	Aspergillus conidia	`WGHSRDE`	G[AST]XVS	Aspergillus hyphae	`GPIVSFG`
		`WGHSRDE`			`AGTGVS-G`
		`WGHSRDE`			`GGSKVSA`
		`WGHSRDE`			`RNGSHVS`
		`WGHSRDE`			`GAAVSIL`
		`WGHSRDE`			`GAVVSVD`
		`WGHSRDE`	[GD][RS]LG	Aspergillus hyphae	`GGRLGPF`
		`WGHSRDE`			`GGRLGPF`
		`WGHSRDE`			`GDRLPHF`
		`WGHSRDE`			`RSIGRLG`
		`WGHSRDE`			`PAEGSLG`
		`WGHSRDE`	LhRSp	Aspergillus hyphae	`VLLRSSG`
		`WGHSRDE`			`FLLRSND`
		`WGHSRDE`			`RLWRSTG`
		`WGHSRDE`			`VPLSRST`
		`WGHSRDE`	GXRS	Aspergillus hyphae	`IGGRSTL`
		`SGGRRLE`			`ARIGSRS`
		`SGGRRLE`			`GVRSSSA`
		`SGGRRLE`	R[GST]XXWS	Aspergillus hyphae	`RGFAWSP`
		`SGGRRLE`			`RSERWSG`
A[T]PS	Aspergillus conidia	`LLSATPS`			`RTSGWSE`
		`LLSATPS`	ASV	Aspergillus hyphae	`DMLGASV`
		`LLSATPS`			`ASVHTLD`
		`LLSATPS`			`ASVARII`
		`LLSATPS`	AAG	Aspergillus hyphae	`AAGIGGD`
		`LLSATPS`			`DMAAGMA`
		`LLSATPS`			`WGVAAGG`
		`LLSATPS`	G[DQ]XSXV	Aspergillus hyphae	`GDASGVV`
		`LLSATPS`			`GDGSWVG`
		`LLSATPS`			`GGQHSEV`
		`LLSATPS`	LXGR	Aspergillus hyphae	`LLGRISR`
GG-R[GR]L[DE]	Aspergillus conidia	`GGVRGLD`			`LLLGRFA`
		`GGVRGLD`			`EIALRGR`
		`GGVRGLD`	LXS[NSK]S	Aspergillus hyphae	`DLTSNSR`
		`GGVRGLD`			`ALKSSSN`
		`GGVRGLD`			`RPLGSKS`
		`GGVRGLD`	Others	Aspergillus hyphae	`VGTDYVG`
		`SGG-RRLE`			`VLNDLVA`
		`SGG-RRLE`			`RMFNSVA`
		`SGG-RRLE`			`WMFGSGA`
		`SGG-RRLE`			`NPGYWGN`
		`TGG-RVLS`			`YYPGYDA`
GARA[DS][AG][As]	Aspergillus conidia	`GARASGS`			`GALGDXX`
		`GARASGS`			`QALGQLD`
		`GARASGS`			`IGRAPQM`
		`GARASGS`			`GRFMQLL`
		`GARASGS`			`GLDTRGG`
		`ARADAAT`			`GAGHAGG`
		`ARADAAT`			`IHEYGVQ`
GSS	Aspergillus conidia	`ATRDGSS`			`TRYEVGV`
		`ATRDGSS`			`GRAFTSI`
		`ATRDGSS`			`RYLRAVT`
		`ATRDGSS`			`PIG--LGLV`
		`SGSSIDQ`			`GPHKGLV`
		`GSSSGNF`			`LGVSLIA`
Others	Aspergillus conidia	`DANRVGG`			`YFGVSRS`
		`GAIRSGL`			`PFVAYDP`
		`GWQGFGS`			`GSPVAYS`
		`SGEVGRG`			`KSSTTAS`
		`GGWGPGS`			`STTRENH`
		`AGSGLSN`	No apparent patterns (RS motif)	Aspergillus hyphae	`RWSEKMR`
`AGGQKSI`	`RIWSDYR`		No apparent patterns (RS motif)
		`TGGRVLS`			`LLYSNGR`
		`VGVRPDS`			`LSRDRVR`
		`GWHPGQE`			`RWTLAGC`
		`XXHAGQA`			`TGRRHSV`
		`EGLRRSA`			`XKXRELX`
		`EYLGLAR`			`WRQSTEG`
No apparent pattern (RS motif)	Aspergillus conidia	`RESVRDR`			`FHPLRDG`
No apparent pattern (RS motif)	`NEYSRPG`	`FDWQGRV`
		`XAGPXRT`			`GQDTGSL`
		`TVDSARS`			`QPREFEL`
		`-DGRGMA`			`XCDSSVS`
		`XTRSGHX`			`YNSLVTH`
		`LEWTGLD`
		`ATGTHGP`

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Peptide sequences preferentially isolated from the surface of Aspergillus conidia and hyphae were aligned to obtain motifs shared between different peptides (Clustal W program; European Molecular Biology Laboratory). The software registers sequence identities and similarities among multiple peptide sequences and aligns the sequences by placing peptides with the most similarity or identity next to one another. Similarity between peptides at the level of amino acid sequence is presented by boldface type.

TABLE 2.

Candidate proteins mimicked by selected peptide motifs after selection for AF293 conidia^a

Extended motif	Human protein containing the motif	Protein description	Accession no.	Alignment	Homology score^b
WGHSRDE	Collagen VI	ECM protein	NP_001839	`WGSRD`	25.8
DLGRVGG				`DLGVG`	25.0
ISKQGGA				`ISQGA`	23.1
GAIRSGL				`AI+GL`	22.3
GRGVEGS				`RGV+G+`	22.3
TAADARA				`TAAD`	21.6
GSSSGNF				`GS+SGNF`	20.8
GWQGFGS				`G+QGGS`	20.8
RESVRDR				`+++RDR`	18.9
QDISSGS				`++SSG+`	18.5
QDISSGS	Phosphacan	Proteoglycan	AAC52383	`DSSGS`	24.6
VGVRPDS				`VGVDS`	23.9
RALGLYE				`RALL+E`	22.3
WGHSRDE				`HSR+E`	22.3
LLSATPS				`S+TPS`	21.9
NSLGSGA				`SLSG+`	21.9
SGSARAG				`SS+AG`	21.9
LLSATPS	Versican	Proteoglycan	AAC24358	`LLSTPS`	29.6
ATRDGSS				`ATRGS+`	26.2
TAADARA				`AA+AR`	22.3
GRDSPSA				`+LGSG`	21.9
NSLGSGA				`SPSA`	21.6
SSGDRTA				`SGDR`	21.6
DANRVGG				`NRVG`	21.6
LLSATPS	Syndecan	Proteoglycan	NP_037158	`LL+ATP+`	26.6
SSGDRTA				`+SG+TA`	22.7
GGVRGLD				`GG+GL`	20.8
LLSATPS	Heparan sulfate receptor	Proteoglycan receptor	AAA37087	`LL+ATP+`	26.6
TAADARA				`T+DA+A`	21.9
GGVRGLD				`GG+GL`	20.8
LLSATPS	DSD-I proteoglycan	Proteoglycan	CAB41976	`S+TPS`	21.9
GRDSPSA				`GRDS`	21.6
GRGVEGS				`VEGS`	21.6
GWQGFGS				`GFGS`	21.6
GARASGS	T-cell receptor β-chain	Cellular immune response	AAD29501	`ARASG`	25.8
SSGDRTA				`SGDTA`	21.2
RALGLYE				`RAG+E`	20.4
GARASGS	Cytokeratin XIV, XVIII	Keratin-like molecule	XP_094275	`GARSGS`
GARASGS	Tumor necrosis factor ligand superfamily IX	Membrane receptor	NP_003802	`GARASGS`	29.6
ELRRGGS				`ELRRGGS`	24.3
NEYSRPG				`YSPG`	20.8
GARASGS	Nebulin-related anchoring protein	Anchoring protein	AAL99185	`ARASG`	25.8
RESVRDR				`+SVRD`	22.3
ELRRGGS	Lymphocyte α-kinase	Protein kinase	NP_079420	`ELRRGG`	30.0
GSSSGNF				`GSSG+`	21.9
NSLGSGA				`NSLG+`	21.9
QDISSGS				`SSGS`	21.6
VGVRPDS				`GVRD`	20.8
GAIRSGL	1, 4-alpha-glucan branching enzyme	Biosynthetic enzyme	JX0243	`GA+SG`	22.3
DLGRVGG				`GRVG`	21.6
TVDSARS				`VDAS`	21.6
GGWGPGS				`GGWGS`	20.8
GPRSYEN				`PRYE`	20.8
SSGDRTA	Cartilage aggregating proteoglycan	Proteoglycan	CAA42701	`SSGDRTA`	30.0
SLVRGGT				`SLVR`	21.6
SSGDRTA	IgG heavy-chain variable region	Humoral immune response	CAC29392	`SSGDRT`	30.0
GRGVEGS				`G+G+E`	18.5
GSSSGNF				`+SSG+`	18.1
RESVRDR				`+SV+R`	18.5
GGWGPGS	Properdin precursor (factor P)	Complement component	Q64181	`G+GWG`	21.6
SGFGQWG				`G+GWG`	21.6
TAADARA				`TDRA`	20.8
GGWGPGS	Thrombospondin I	ECM protein	NP_035710	`GGWGPS`	29.3
GDGRGMA				`GDGRGA`	28.9
GRGVEGS				`GRGVE`	25.8
AGGQKSI				`AGQKSI`	24.6
QDISSGS				`QD+SS`	22.3
RESVRDR				`R+VDR`	21.6
SGGRRLE				`GRRL`	21.6
TVDSARS				`TVDS`	21.6
GGWGPGS	Chemokine ligand I	Membrane receptor	NP_002987	`WGPGS`	25.8
EGLRRSA				`EGLRRS`	24.6
GARASGS				`ASGS`	21.6
GRGVEGS				`GVGS`	21.2
LLSATPS				`LTPS`	21.2
RALGLYE	C₉ molecule	Complement component	AAA51889	`GLYE`	21.6
ELRRGGS				`LRGG+`	20.8
SLVRGGT	C₈-β molecule	Complement component	BAC41370	`LVRGGT`	30.0
ELRRGGS				`LRGG+`	20.8
GRGVEGS				`GGV+GS`	20.8
LLSATPS	Major histocompatibility complex class I heavy chain	Major histocompatibility complex	AAG02528	`LLTPS`	25.4
TAADARA	Major histocompatibility complex class I heavy chain	Major histocompatibility complex	`TAA++A`	22.3
ISKQGGA				`I+KQGG`	20.0
IGRAPQM	Immunity-associated protein	GTP immune-associated nucleotide	NP_787056	`RAPQM`

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For similarity searches, peptides recovered from panning were analyzed with BLAST (National Center for Biotechnology Information [NCBI]) searches for similarity to known human proteins in the NCBI database. Motif regions of 100% identity between peptides selected from panning and the candidate protein are shown. Conserved amino acid substitutions are indicated by plus signs. ECM, extracellular matrix.

A complete seven-amino-acid match gave a homology score of 34, and a complete six-amino-acid match gave a homology score of 30. The score matrix was based on modified BLOSUM62 to minimize the problem that some rare amino acids in the BLOSUM62/PAM30 matrix are heavily weighted, which tends to overpower the perfect matches of four- to five-amino-acid stenches.

TABLE 3.

Candidate proteins mimicked by selected peptide motifs after selection for AF293 hyphae^a

Extended motif	Human protein containing the motif	Protein description	Accession no.	Alignment	Homology score^b
GGRLGPF	Collagen I-VII, IX-XI	ECM proteins	NP_031763	`GRLGP`	25.8
GSVGTGA				`GVGTGA`	25.4
AILSVGL				`ILSVL`	24.3
VGVEYRT				`VGEYRT`	24.3
GGAHGAG				`GGAAG`	24.3
GDGSWVG				`GGSVG`	23.5
IIRAVSA	Collagen XIV-XVII, XIX	ECM proteins	AAB19038	`II+VS`	22.3
GGRLGPF				`GLGP+`	21.9
GLSGEAP				`GLGE`	21.6
GSVGTGA	Collagen XXII, XXIV	ECM proteins	NP_690850	`GSVGTG`	25.4
GPIVSFG				`GPI+FG`	25.0
GGRLGPF				`G+LGP`	21.9
GGRLGPF	Thrombospondin I-V	ECM proteins	NP_035710	`GGRLGF`	28.9
ADYGPYY				`ADGPY`	24.6
GGAAVGW				`GAVG+`	21.9
RGRLAIE				`RGLA+E`	21.9
GVRSSSA				`G+SSS`	21.6
NGWYGPN				`NG+GP`	21.6
GDGSWVG				`DGSW`	21.6
GGRLGPF	Nicein	Laminin-like protein	NP_061486	`GGRLGF`	28.9
RGRLAIE				`RGLA+E`	21.9
GQDTGSL				`GQDS`	21.2
GGAHGAG				`GGAGG`	20.8
QPREFEL				`R+F++`	15.4
GGAHGAG	Laminin V	ECM protein	AAM03454	`GGAHGAG`	22.3
GRFMQLL				`+FMQL`	21.2
GLSGEAP				`LSEA`	20.8
GGSKVSA				`GGSA`	20.0
GGRLGPF	Laminin gamma-III chain	ECM protein	AAD29851	`RLGPF`	25.8
QPREFEL				`REF+L`	22.3
GGAHGAG				`GGAHGAG`	22.3
AMGAAMD				`A+GAAD`	21.6
RGRLAIE				`GLA+E`	21.6
RPLGSKS				`RLGS+`	21.6
LSRDRVR				`SR+RVR`	19.2
GSVGTGA	Macrophage scavenger receptor-like I	Cellular immune response	NP_057324	`GSGTG`	25.4
GGRLGPF	Macrophage scavenger receptor-like I	`GGRLGP`	22.7
DLQGLAQ				`+LQGL`	21.9
HNERTTS				`RTTS`	21.6
RSIGRLG				`S+GLG`	21.6
GGRLGPF	T-cell receptor β-chain	Cellular immune response	AAC69961	`GRLGP`	25.8
LLLGRFA				`LLLGA`	24.3
RSIGRLG				`RSGRLG`	23.9
VSAGLMD				`+AGL+D`	22.3
PIGLGLV				`+GGLV`	21.9
DLTHVSA	Basilin	ECM protein	AAM53532/	`DLTHVS`	30.0
AILSVGL			NP_660140/	`A+LSV`	22.7
DLQGLAQ			Q8K482	`DLQL+`	21.6
GQDTGSL				`G+GSL`	21.6
GTSRWLR	Agrin	Basic membrane component	NP_067617	`T+RWLR`	26.2
RYLRAVT				`YLAVT`	25.8
VPLSRST				`+PLSST`	25.4
GLSGEAP				`LSGEAP`	25.0
VVGSADG				`VSADG`	25.0
VLLRSSG				`V+LRS+`	22.3
XCDSSVS	NEPH2 immunoglobulin domain	Protein-protein interaction molecule	XP_235986	`CDSSVS`	30.0
GAAVSIL				`GA V+ L`	21.9
GRAFTSI				`G+FT+I`	21.9
NGWYGPN				`NG+YN`	21.6
FHPLRDG				`LRDG`	21.6
XCDSSVS	Cartilage homeoprotein I	ECM protein	Q91574	`CDS+VS`	26.2
VVGSADG				`GSA+G`	21.9
GLSGEAP				`LSGA`	21.6
GVRSSSA				`RSSS`	21.6
IGGRSTL				`+GGTL`	21.6
NSSSKLA				`SSSK`	21.6
GGSKVSA				`GGSV++`	21.6
HNERTTS				`H+RTS`	21.6
VVGSADG	NEPH1 immunoglobulin domain	Protein-protein interaction molecule	AAK00529	`VVGSAD`	30.0
DVSVVAG				`D+VVAG`	25.4
VLLRSSG				`VLLSG`	25.0
GRAFTSI				`GRFT`	21.6
HNERTTS				`ERTS`	21.6
RGRLAIE				`RGRL`	21.6
ISTFARG	T-cell receptor α-chain	Cellular immune response	CAB92465	`ISTFRG`	30.0
VGVEYRT	ICAM V	Intercellular adhesion molecule	AAH26338	`VGVEYR`	30.0
GTSRWLR				`GTRLR`	25.0
GGAHGAG				`GGAGA`	25.0
PIGLGLV				`PGLGL`	24.6
AILSVGL				`A+L++GL`	23.5
VGVEYRT	Telencephalin	Intercellular adhesion molecule	NP_032345	`VGVEYR`	30.0
GTSRWLR				`GTRLR`	25.0
PIGLGLV				`PGLGL`	24.6
GGAAVGW				`GGAAG`	24.6
AILSVGL				`A+L++GL`	23.5
LLGRISR				`LLG++R`	22.3
NPGYWGN	Immunoglobulin alpha VH4	Humoral immune response	AAG21420	`PGYWG`	25.8
NPGYWGN	Immunoglobulin heavy chain, VH3 family	Humoral immune response	AAQ05568	`PGYWG`	25.8
IGGRSTL	Immunoglobulin heavy chain, VH3 family	`GRTL`	21.6
NPGYWGN	Iron transporter	Molecule transporter	NP_011072	`NPGYW`	25.8
YFGVSRS				`Y+GS+S`	23.5
VSAGLMD				`+SAGL`	22.7
HNERTTS				`E+TTS`	21.9

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For similarity searches, peptides recovered from panning were analyzed with BLAST (NCBI) searches for similarity to known human proteins in the NCBI database. Motif regions of 100% identity between peptides selected from panning and the candidate protein are shown. Conserved amino acid substitutions are indicated by plus signs. ECM, extracellular matrix.

A complete seven-amino-acid match gave homology score of 34, a complete six-amino-acid match gave homology score of 30, and the score matrix was based on modified BLOSUM62 to minimize the problem that some rare amino acids in the BLOSUM62/PAM30 matrix are heavily weighted, which tends to overpower the perfect matches of four- to five-amino-acid stenches.

Specifically, a peptide motif (sequence CWGHSRDEC) with high similarity to collagen VI was evaluated further as a potential ligand on the surface of Aspergillus fumigatus AF293 conidia. This most commonly recovered CWGHSRDEC motif showed enrichment during successive rounds of panning (i.e., from 9.7% in the third round of selection to 28.3% in the fourth round of selection) and was found to reside on the surface of the von Willebrand domain I of collagen VI (http://www.ncbi.nlm.nih.gov/structure/). The second-most-common conidium-associated recovered CLLSATPSC motif also showed enrichment during successive rounds of panning (i.e., from 3.3% in the third round of selection to 21.7% in the fourth round of selection). In addition, other molecules of the extracellular matrix (e.g., proteoglycans) and immune system effector molecules (e.g., complement components) were also identified as potential ligands on the surface of AF293 conidia (Table 2). More diversity inpeptide motifs was observed in the results for potential ligands binding to the surface of AF293 hyphae (Table 3). Again, a variety of molecules of the extracellular matrix including collagen types I to VII, IX to XI, and XV; thrombospondins I to V, laminin V; basilin (an elastin-like protein); agrin (a component of the lung and kidney basal membrane); intercellular adhesion molecule (ICAM) V; and receptors of effector immune system cells such as T lymphocytes and macrophages were identified as potential Aspergillus hyphal ligands (Table 3). As shown here, phage selection for Aspergillus conidia and hyphae yielded peptides that mimic several human proteins known to be present in the lungs (6).

Validation of phage binding to conidia and hyphae of Aspergillus and other molds.

We then characterized the binding of CWGHSRDEC-displaying and CGGRLGPFC-displaying phage, the phage clones most frequently recovered from AF293 conidia (Table 2) and hyphae (Table 3) and analyzed the degree of their binding to AF293 conidia and hyphae relative to control phage by the BRASIL method. Satisfyingly, we found that binding of the CWGHSRDEC-displaying and CGGRLGPFC-displaying phage recovered from AF293 conidia and hyphae was significantly higher than control in all experimental conditions tested (P < 0.01) (Fig. 1A and B).

FIG. 1. — Validation of phage binding to conidia and hyphae of *Aspergillus* and *Rhizopus* clinical isolates. (A) Binding of the CWGHSRDEC-displaying (relative to control) phage to conidia of AF293 using different TU of the phage in independent experiments (10⁶ to 10¹⁰ TU). (B) Binding of the CGGRLGPFC-displaying (relative to control) phage to hyphae of AF293 using different TU of the phage in independent experiments (10⁶ to 10¹⁰ TU). (C) Binding of the CWGHSRDEC-displaying (relative to control) phage (titer, 10⁹ TU) to conidia of patient-derived *Aspergillus* and *Rhizopus* isolates. (D) Binding of the CGGRLGPFC-displaying (relative to control) phage (titer, 10⁹ TU) to hyphae of patient-derived *Aspergillus* and *Rhizopus* isolates. Values are averages from three independent experiments after one round of biopanning; bars represent standard deviations. (No significant binding of the CWGHSRDEC-displaying phage was observed relative to control phage to conidia of the *Aspergillus flavus* isolate AF122 [data are not shown in Fig. 1C)] and no significant binding of the CGGRLGPFC-displaying phage was observed relative to control phage to hyphae of the *Aspergillus flavus* isolate *AF117* [data are not shown in Fig. 1D]).

We next tested whether this binding would also be seen in other patient-derived mold isolates. Again, CWGHSRDEC-displaying phage recovered from conidia of different Aspergillus and Rhizopus isolates was significantly higher than that of a control (P < 0.01), with the exception of conidia of Aspergillus flavus A122, where no significant binding of the CWGHSRDEC-displaying phage was observed relative to control phage (Fig. 1C). Similarly, the CGGRLGPFC-displaying phage recovered from hyphae of different Aspergillus and Rhizopus isolates was significantly higher than control (P < 0.001), with the exception of hyphae of Aspergillus flavus A117, where no significant binding of the CGGRLGPFC-displaying phage was observed relative to control phage (Fig. 1D). The reason(s) for the lack of binding in either A117 or A122 isolates remain(s) an open question; future studies focusing on larger number of isolates of each Aspergillus species should address the possibility of Aspergillus species-specific differences in phage binding.

These results show that the two most commonly recovered peptide-displaying phage from AF293 conidia and hyphae also bind to the surface of conidia and hyphae of a variety of other clinical mold isolates recovered from cancer patients, suggesting that this binding is not likely strain specific.

A selected binding peptide targets Aspergillus fumigatus hyphal elements on lung tissue recovered from mice with invasive pulmonary aspergillosis.

Having characterized the in vitro binding of CGGRLGPFC-displaying phage to hyphae of various mold isolates, we then assessed whether this phage clone also binds to Aspergillus hyphae recovered from mice with invasive pulmonary aspergillosis. We focused on studying the binding of hypha-associated peptides to mouse lung tissue with invasive pulmonary aspergillosis because Aspergillus hyphae are the predominant fungal forms that are seen during invasive growth in tissues, although Aspergillus conidia and conidiophores can occasionally be seen (4, 20). To that end, we performed immunohistochemistry and immunofluorescence studies of immunosuppressed mice infected with Aspergillus fumigatus.

Consistently, CGGRLGPFC-displaying phage binding to Aspergillus hyphae was strong and present in all tested sections of lung tissue in the mice with invasive pulmonary aspergillosis (Fig. 2C and F). In contrast, control phage binding to AF293 hyphae was either absent or barely detectable relative to CGGRLGPFC-displaying phage binding in the lungs (Fig. 2B and E). IgG did not immunoreact with Aspergillus hyphae that was previously coincubated with CGGRPGPFC-displaying phage (Fig. 2A and D).

FIG. 2. — Immunohistochemistry analysis following phage overlay assays on representative lung tissue sections from mice with invasive pulmonary aspergillosis. (A and D) Results following addition of CGGRLGPFC-displaying phage and rabbit IgG. (B and E) Results following addition of control phage and anti-phage antibody. (C and F) Results following addition of CGGRLGPFC-displaying phage and anti-phage antibody. Note the stronger signal on *Aspergillus* hyphae than the signal generated by the addition of control phage. Bar, ∼15 to 20 μm.

By using immunofluorescence, CGGRLGPFC-displaying phage bound specifically to AF293 hyphae on lung tissue sections from mice with invasive pulmonary aspergillosis (Fig. 3). The possibility that this hypha-specific signal was due to anti-phage or anti-rabbit secondary antibody cross-reactivity with Aspergillus hyphae was excluded by the addition of anti-phage antibody without previously adding CGGRLGPFC-displaying phage and by the addition of anti-rabbit secondary antibody without previously adding anti-phage antibody, respectively.

FIG. 3. — Immunofluorescence studies of representative lung tissue sections from mice with invasive pulmonary aspergillosis. (A, C, and E) Hypha-specific signal achieved following addition of CGGRLGPFC-displaying phage and anti-phage antibody. (B, D, and F) Respective DAPI counterstaining. Panels E and F represent a higher magnification (×730) of panels C and D, respectively (×292). Bar, ∼25 to 30 μm.

Thus, the results of immunostaining that demonstrate specific and reproducible binding to the mouse lung tissue with invasive pulmonary aspergillosis are consistent with our in vitro findings that CGGRRGPFC-displaying phage binds to the surface of Aspergillus hyphae.

DISCUSSION

In the present study, we used a combinatorial peptide approach for selecting cell surface motifs from phage display libraries for an infectious disease application. As a proof of concept, we used the BRASIL method to screen for enriched peptides on the surface of conidia and hyphae of the medically significant fungus Aspergillus fumigatus. To isolate shared and different motifs between both developmental programs of growth of Aspergillus fumigatus, we performed parallel screens of a phage display library with both conidia and hyphae.

We identified several peptide motifs after phage selection for Aspergillus conidia and hyphae that mimic human proteins of the extracellular matrix such as various collagen subtypes and proteoglycans. These proteins, which are quite abundant in the human airways and lung parenchyma (6), likely play a significant role in the initial attachment of Aspergillus conidia to airway and lung tissue molecules and in the subsequent invasion of Aspergillus hyphae into the lungs. Of interest is also the identification of hypha binding peptide motifs that mimic ligands associated with effector immune cells such as macrophage and T-lymphocyte receptors as potential ligands on the Aspergillus hyphal surface, since macrophages and T lymphocytes are critical components of the human immune response against invading Aspergillus (4, 20). Nevertheless, the true identity of the proteins mimicked but those targeting peptides remains an open question that should be the focus of future studies.

The fact that we did not find sequences from the initial rounds of selection that were identical between Aspergillus conidia and hyphae is probably explained by the fact that we sequenced the DNA corresponding to peptide inserts of approximately 50 to 70 randomly picked phage clones for Aspergillus conidia and hyphae. Thus, if we had sequenced more phage clones, it is very likely that we would have found similar sequences from the selection of Aspergillus conidia and hyphae. This assumption is based on other experiments that demonstrated that representative conidium-associated displaying phage (e.g., CWGHSRDEC and CLLSATPSC) also bound (even though less avidly) to Aspergillus hyphae (data not shown). Similarly, representative hypha-associated displaying phage (e.g., CGGRLGPFC and CIIRAVSAC) also bound (although less avidly) to Aspergillus conidia (data not shown). We believe that this phenomenon may reflect common or ubiquitous binding sites on the surface of Aspergillus conidia and hyphae and is likely explained by their common origin, since Aspergillus hyphae germinate from conidia.

The BRASIL method has been widely utilized for the selection of specific ligands because of the intrinsic ability of this methodology to minimize background phage binding (10). Moreover, the system is well suited for preclearing of the library before one selects peptides with the desired specificity. This is an important step, because it decreases the chances of isolating nonspecific binding phage, as these are more than likely to get precleared before the specific selection starts.

It is worth mentioning also that successful phage display screenings do not always result in substantial enrichment from round to round. This is a positive indication that is often utilized as a parameter for prioritizing peptide leads (10). Furthermore, differences in transducing unit counts are a strong indication of specificity and they do not necessarily (or usually) reach log-scale differences to be considered highly selective. Moreover, the BRASIL technology often results in the detection of binding events that are quite robust, given the inherent stringency of the assay.

However, it is the extensive validation at the level of each individual phage that determines the level of certainty of how selective and specific a given ligand is and in recognizing a protein target. Validation at the individual phage level is required, and in our case, it stood the test under well-controlled experimental conditions. The alignment analysis of the peptides suggested that the binding sites in question might well be conformationally defined, since the exact linear amino acid primary sequence of any given peptides was not enriched during the screening. However, we did find residues of the peptides with homology scattered throughout the isolated sequences. Of note, this is a relatively common observation in phage display random peptide screenings (3, 9, 23, 34).

Detection of early invasion by Aspergillus hyphae is the focus of early diagnostic and therapeutic strategies for invasive aspergillosis (17, 20). Therefore, we examined whether the CGGRLGPFC-displaying phage clone, the most commonly recovered peptide-displaying phage from AF293 hyphae, also binds to the surface of hyphae of other patient-derived Aspergillus isolates in vitro. In fact, we found that the CGGRLGPFC-displaying phage bound to 8 (89%) of 9 Aspergillus isolates tested, suggesting that CGGRLGPFC binding is not strain specific. Also of interest is that CGGRLGPFC-displaying phage bound to hyphae of both Rhizopus isolates tested; Rhizopus is a rare, yet emerging, opportunistic mold that shares common pathogenic and clinical features with Aspergillus (18, 20).

To extend this in vitro observation that CGGRPGPFC-displaying phage binds consistently to Aspergillus hyphae also in vivo, we sought to determine whether the CGGRLGPFC-displaying phage binds to Aspergillus hyphal elements on lung tissue sections from mice with invasive pulmonary aspergillosis. By using immunohistochemistry analysis, we found that CGGRLGPFC-displaying phage clone binds reproducibly to hyphae in contrast to control phage. Similarly, by using immunofluorescence we found a hypha-specific signal of the CGGRLG PFC-displaying phage in all histopathology sections tested from mice with invasive pulmonary aspergillosis.

Our findings have potential therapeutic implications. Early invasion by Aspergillus hyphae in the lung parenchyma is frequently missed by even the most sensitive available radiographic methods, such as high-resolution computed tomography of the lungs, which typically has a threshold of detection of lesions of ≥1 mm (12). Thus, identification of hypha-specific peptides may lead to new immunolabeling radiographic approaches for detection of early invasion of Aspergillus hyphae in the lungs.

Finally, antifungal drug efficacy against Aspergillus species in vivo is suboptimal, and this is partially explained because Aspergillus is an angiotropic mold that invades blood vessels, resulting in tissue infarcts and low tissue perfusion (31). By detecting Aspergillus lesions early before the formation of such grossly hypoperfused lesions and, ideally, by coupling an antifungal with hypha-specific peptides, it might be possible to enhance the activity of antifungal agents in vivo by increasing local and early delivery to the site of infection. Targeted delivery of cytotoxic drugs (2), proapoptotic peptides (8, 16), cytokines (5), metalloprotease inhibitors (15), genes (11, 25), or liposomes (30) to tumor vasculature receptors has resulted in marked therapeutic efficacy in tumor-bearing mice and is the subject of current research. Our study adds invasive aspergillosis as an example of a prototypic infectious disease that can be approached in a similar way.

We believe that a peptidomimetic approach based on peptides holds more promise as a delivery tool than antibodies for several reasons. As opposed to antibodies, which can be difficult and expensive to produce on a large scale, small molecules such as peptidomimetics offer great stability (24). Moreover, they are suitable for the simple design of targeted compounds by Merrifield synthesis, without the need for chemical conjugation (24). Also, although peptides often show lower-affinity binding constants than antibodies, their tissue penetration profile may compensate for it (24). Finally, one must be careful when extrapolating data derived from comparing antibodies to ligand peptide-based binding, particularly in the context of antibody versus peptide phage display selection. It may be true that inhibitory peptides isolated by phage display are unlikely to serve as agents per se but (i) they may form the basis for rational peptidomimetic development and (ii) they may serve as tools for finding targets against which a good antibody may also be a suitable choice for translational applications.

Direct comparison of peptides and antibodies targeting vascular receptors has been performed for both CD13-targeting ligands (28) and aminopeptidase A-targeting ligands (22). In both instances, peptides or antibodies were able to home to tumors by binding to these vascular addresses and to functionally block proangiogenic activity of these proteins in the context of tumor vasculature. It is possible that lower-affinity ligands might preferentially accumulate at sites in which the target is highly expressed. As opposed to higher-affinity ligands, such as antibodies, peptides bind to target receptors in a manner that is directly dependent on receptor expression levels. However, these two approaches are nonmutually exclusive and may actually be complementary: as discussed above, peptides may be useful for identifying targets for antibody-based therapy and also for rationally developing peptidomimetic drugs. Hence, future work should compare and contrast different classes of Aspergillus ligands. Finally, selection of an antibody phage display library (rather than a peptide library) by using the BRASIL method may be possible in future studies. If so, selection of targeting antibodies may be feasible.

In conclusion, we show that a peptide-displaying phage binds to the invasive hyphal form of Aspergillus fumigatus both in vitro and to lung tissue recovered from mice with invasive pulmonary aspergillosis. Ligand-directed targeting of this opportunistic mycosis may lead to new imaging and therapeutic interventions in invasive aspergillosis. Finally, these results may become useful in studying host immune responses against Aspergillus by focusing on fundamental differences in ligand binding sites between effector immune cells of the innate immunity (e.g., resting lung macrophages) and those of the adaptive immunity (e.g., lymphocytes).

Acknowledgments

We thank George Z. Rassidakis from The University of Texas M. D. Anderson Cancer Center for his assistance in the immunohistochemistry and immunofluorescence studies.

This work was supported in part by the M. D. Anderson Faculty E. N. Cobb Scholar Award Research Endowment and the M. D. Anderson Cancer Center Core Grant (CA16672) from the University of Texas to D.P.K. and from the Gilson-Longenbaugh Foundation to W.A. and R.P.

Editor: T. R. Kozel

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PERMALINK

Development of a Ligand-Directed Approach To Study the Pathogenesis of Invasive Aspergillosis

Michail S Lionakis

Johanna Lahdenranta

Jessica Sun

Wei Liu

Russell E Lewis

Nathaniel D Albert

Renata Pasqualini

Wadih Arap

Dimitrios P Kontoyiannis

Abstract