Dermatophytic with antiinfective potential

Shunyi Zhua,1, Bin Gaoa, Peta J. Harveyb, and David J. Craikb

aGroup of Animal Innate Immunity, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, 100101 Beijing, China; and bInstitute for Molecular Bioscience, University of Queensland, Brisbane Queensland 4072, Australia

Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved April 6, 2012 (received for review January 25, 2012)

Fungi are a newly emerging source of with micasin) from the dermatophytic Microsporum canis using therapeutic potential. Here, we report 17 new fungal defensin-like the synthetic peptide. Our results show that fungal genomes are a peptide (fDLP) genes and the detailed characterization of a corre- source of antiinfective therapeutic agents that can be rapidly sponding synthetic fDLP (micasin) from a dermatophyte in terms characterized by an integrated computational and experimental of its structure, activity and therapeutic potential. NMR analysis approach. The potential applications of dermatophytes in devel- showed that synthetic micasin adopts a “hallmark” - oping drugs against skin-derived bacterial infections (e.g., Staphy- stablized α-helical and β-sheet fold. It was active on both Gram- lococcus aureus and P. aeruginosa) are highlighted. positive and Gram-negtive , and importantly it killed two clinical isolates of methicillin-resistant Staphylococcus aureus Results and the opportunistic Pseudomonas aeruginosa at low Fungal Genomes Are a New Source of Defensin-Like . From micromolar concentrations. Micasin killed approximately 100% of recently sequenced fungal genomes, we identified 17 DLP genes. treated bacteria within 3 h through a membrane nondisruptive These DLPs can be grouped into four different families based mechanism of action, and showed extremely low hemolysis and on their sequence similarity, three of which are classified into high serum stability. Consistent with these functional properties, known families (fDEF1, fDEF2 and fDEF5) (12) (Fig. 1). In micasin increases survival in mice infected by the pathogenic comparison with fDEF1, fDEF2 has a smaller, less anionic pro- bacteria in a peritonitis model. Our work represents a valuable peptide, whereas the precursors of fDEF5 and the family fDEF7 approach to explore novel peptide antibiotics from a large resource lack a propeptide. Peptides in fDEF7 have a unique structural of fungal genomes. characteristic in that their amino-termini are longer and rich in histidine compared with other DLPs. ∣ methicillin-resistant Staphylococcus aureus ∣ plectasin ∣ The fDEF1 members belong to ancient invertebrate-type mouse peritonitis model (AITDs) (12) and they all originated from Pezizomy- cota (Ascomycota) classified into three genera: Ajellomyces, ntimicrobial peptides (AMPs), typically 12–80 amino acids in Arthroderma, and Trichophyton (Table S1). Species derived from Asize, are key effectors of innate immunity in multicellular the latter two genera belong to dermatophytic fungi. The mature organisms (1). They comprise the first line of defense to rapidly DLPs share 30% to 60% sequence identity with plectasin, and clear in the early stage of infection through a multi- they all carry net positive charges from þ1.2 to þ5.2 at physio- target mode of action, which may serve to prevent or delay evolu- logical pH (Fig. 1A), providing a possibility to bind polyanionic tion of microbial resistance. Their diverse action modes include bacterial membranes by electrostatic interaction. Despite the disruption of membrane integrity, impairment of nucleic acid overall net positive charge nature, these peptides have a net ne- and protein synthesis, inhibition of chaperone-assisted protein gatively charged N-terminal loop (n-loop), as in the case of folding, interruption of cell-wall biosynthesis pathway, and even plectasin. Aside from six conserved , the fDEF1 family targeting of membrane biogenesis (2–4). Some vertebrate AMPs has three identical residues (Ser17, Gly19, and Gly23, numbered (e.g., human cathelicidins and defensins) have also evolved a mul- according to micasin) whose conservation is across the alignment. tifunctional, immunomodulatory ability in bridging the innate The fDEF2 members belong to classical invertebrate-type defen- and adaptive immune systems (5). sins (CITDs) (Fig. 1B). However, unlike other known CITDs The unique properties of AMPs make them attractive candi- (12), these fungal CITDs carry net charges of −0.6 to −4.8,a dates for the development of antiinfective drugs with reduced characteristic previously observed in the tick scasin-type defen- risk of resistance (1, 6), especially when used in combination. To sins (13). Except labisin in Basidiomycota (Agaricomycotina), date, some such peptides and their derivatives are in clinical trials a sister group to the Ascomycota, all other members come from – (7 9; however, they have proven difficult to reach the market as Ascomycota (Pezizomycotina). Analysis of exon-intron structures systemic (parenteral and oral) therapeutics owing to limitations of the newly discovered DLPs revealed that despite low sequence including nonspecific toxicity, suboptimal efficacy, and lability similarity among families 1, 2, and 7, some of them share a con- to serum proteolysis (7, 8). Studies have shown that when systemi- served phase-0 intron within the α-helical region (Fig. 1), suggest- cally applied, some mammalian AMPs produce side effects, ing common ancestral origin for these DLP families. probably due to their immunomodulatory properties interfering with the normal immune network and inducing an exacerbating inflammatory response. Turning to nonmammalian sources of Author contributions: S.Z. designed research; S.Z., B.G., P.J.H., and D.J.C. performed research; S.Z., B.G., P.J.H., and D.J.C. analyzed data; and S.Z., P.J.H., and D.J.C. wrote

AMPs offers the potential to develop new classes of SCIENCES agents. Plectasin, the first defensin isolated from the saprophytic the paper. Conflict of interest statement: Based on this work, a patent application (“Novel fungus nigrella (Ascomycota: Pezizomycota), is APPLIED BIOLOGICAL fungus-derived antiinfective defensins”) has been filed. of particular interest as it selectively targets Gram-positive bac- teria with particular activity against pneumoniae This article is a PNAS Direct Submission. (10). Its lack of induction of the inflammatory cytokine interleu- Data deposition: The sequences reported in this paper have been deposited in the GenBank database (http://www.ncbi.nlm.nih.gov/) [accession nos. JN014007 (micasin) and kin-8 (IL-8) has also been confirmed (11). Following its discov- JN014008 (micasin-1)]. The atomic coordinates of micasin have been deposited in the ery, plectasin-related peptides in fungi have also been reported Protein Data Bank, www.pdb.org (PDB ID code 2LR5) recently using bioinformatics approaches (12). 1To whom correspondence should be addressed. E-mail: [email protected]. Here, we describe 17 fungal defensin-like peptide (DLP) genes This article contains supporting information online at www.pnas.org/lookup/suppl/ and the detailed characterization of a DLP (herein referred to as doi:10.1073/pnas.1201263109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1201263109 PNAS ∣ May 29, 2012 ∣ vol. 109 ∣ no. 22 ∣ 8495–8500 Downloaded by guest on September 29, 2021 A

Fig. 1. Multiple sequence alignment of fungal DLP fa- milies. (A) fDLP1; (B)fDLP2;(C) fDLP5; (D)fDLP7.Signalpep- tides, propeptides, and ma- ture peptides are boxed in black, red, and green, respec- tively. Cysteines are shadowed in yellow; basic and acidic resi- dues are shown in blue and red, respectively. The pairing pattern of the three disulfide B bridges and secondary struc- tural elements (cylinder: α-he- lix; arrow: β-strand) were extracted from the coordi- nates of plectasin (Protein Data Bank entry 1ZFU). HRR, histidine-rich region. Introns are indicated by arrows or small boxes. a, peptide size; b, identity to plectasin; c,net C charges, estimated at pH 7.0 using protein calculation V3.3 (http://www.scripps.edu/ ~cdputnam/protcalc.html). D Sequences described pre- viously (10, 12) are indicated by an asterisk.

M. canis DLPs. M. canis is the only species whose genome encodes (4061.68 Da) calculated from its primary sequence, indicating two DLPs (micasin and micasin-1) with low sequence similarity. that six hydrogen atoms in the cysteines of the reduced peptide These two DLPs are functional genes, as evidenced by cDNA have been removed when three disulfide bridges are formed. cloning (Fig. 2A; Fig. S1). In a Neighbor-Joining (NJ) tree, We then studied the structural features of micasin by circular micasin-1 is separated as a single clade with a large distance from dichroism and found that it displayed a minimum at 208 nm micasin, whereas micasin and other members constitute another and a maximum at 198 nm (Fig. 2D), demonstrating that it has orthologous clade (Fig. 2B), suggesting that these two M. canis folded into a native-like conformation similar to other peptides DLPs originated by an early gene duplication preceding specia- with a cysteine-stabilized α-helical and β-sheet (CSαβ) fold (14). tion of Pezizomycotina. In the subsequent evolution, the ortho- logue of this gene lost in other fungal lineages. Because of Micasin Adopts a Typical CSαβ Fold. The three-dimensional struc- evolutionary retention in genomes of many fungal species hints ture of micasin was determined by NMR spectroscopy. The 1D functional importance of micasin and its orthologues, we thus se- 1H NMR spectrum of micasin showed good dispersion (<2 ppm) lected micasin as a representative for detailed structural and fun- in the amide region, indicative of a highly structured peptide. tional investigations. Two-dimensional spectra were recorded at temperatures ranging Micasin shares 49% to 62% sequence identity with defensins from 283 to 303 K and a full assignment was obtained, except that from oysters, ticks, scorpions, and dragonflies, with four variable no amide resonances could be detected for Arg30, Ala31 or Thr32 regions located at three loops and the carboxyl-terminus 29 (Fig. S2A). We chemically synthesized the reduced form of mica- at any temperature studied and the amide resonance for Leu sin and then folded it by air oxidation in an alkaline environment. was only observed in spectra obtained at 283 K. This lack of The oxidized product was purified to homogeneity by RP-HPLC, signals suggests that this region of the peptide is highly flexible where it was eluted at the retention time (TR) of 21 min, earlier and its proton resonances are broadened beyond detection. De- than its reduced form (TR of 23.2 min) (Fig. 2C), indicating that termination of translational diffusion coefficients from pulse field more hydrophobic residues are less accessible. To verify disulfide gradient NMR experiments indicate that the peptide is mono- bond formation, we analyzed the sample using MALDI-TOF MS meric under the same conditions as used for the structure calcu- and determined an experimental molecular weight (MW) of lations (Table S2). A summary of nuclear Overhauser effect 4055.7 Da (Fig. 2C), 5.98 Da less than the theoretical MW (NOE) data is displayed in Fig. 3A and Hα secondary shifts used

8496 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1201263109 Zhu et al. Downloaded by guest on September 29, 2021 A B 94 Trirusin 99 Tritosin Trivesin 42 57 Micasin Atesin-1 99 Atesin-2 Fig. 2. M. canis DLPs. (A) Amplification Plectasin of micasin and micasin-1 cDNAs by nested Acasin PCR. Bands corresponding to correct pro- 97 ducts are shown by red arrows. (B) Mica- 87 Adersin-1 sin and micasin-1 stemmed from an early Micasin-1 gene duplication event (indicated by a red circle), as revealed by a Neighbor- 0.1 Joining tree constructed based on amino acid sequences of the fDLP1 family by MEGA. Nodal support was estimated C D using the bootstrap approach with 1000 replicates. The scale bar shows total ami- no acid divergence. (C) Oxidative refold- ing of chemically synthetic micasin. RP- HPLC showing retention time (T R) differ- ence between the reduced (R) and oxi- dized (O) peptides. Inset, MALDI-TOF MS of the oxidized peptide. (D) The CD spec- trum of micasin, measured at 25 °C from 260 to 190 nm by using a quartz cell of 1.0-mm thickness. Data were collected at 1-nm intervals with a scan rate of 50 nm∕ min.

to locate elements of secondary structure (15) are provided ulfide connectivities (Cys4-Cys25, Cys11-Cys33, Cys15-Cys35) were in Fig. S3. fully consistent with the NOE data and were included as re- A number of resonances were observed for more than 20 h straints in the structure calculations. The resulting family of struc- after the peptide was dissolved in deuterated water. This finding tures (Fig. 3B) had good structural and energetic statistics, as strongly suggests that these 10 amide protons (Cys11, Ala13, shown in Table S3. A ribbon representation of the secondary His14, Cys15, Leu16, Ser17, Ile18, Phe24, Ala26, and Thr34) are structures is shown in Fig. 3C. Analysis of the structures with involved in significant hydrogen bonds. The three-dimensional PROCHECK (16), excluding the undefined region Leu29-Thr32, structure of micasin was calculated with a total of 265 distance showed that 100% of the residues were in the most favored or restraints, 21 dihedral angle restraints, and 11 hydrogen bond additionally allowed regions of the Ramachandran plot. Analysis restraints using a simulated annealing protocol in CNS. The dis- of the structures with PROMOTIF (17) identified an α-helix D Fig. 3. Three-dimensional A structure of micasin. (A) Summary of NOE data. The thickness of the bars indi- 37 18 R20 K cates the intensity of the d I NN(i, i+1) NOEs; slowly exchanging dαN(i, i+1) 13 amide protons are denoted dβN(i, i+1) A with black circles. (B)A dNN(i, i+2) family of 20 lowest energy dαN(i, i+2) 10 structures superimposed dα E N(i, i+3) over the backbone atoms of dα 2 N(i, i+4) F residues 1–27 and 33–38. (C) dαβ (i, i+3) A ribbon representation A31 28 with disulfide connectivities P shown in ball and stick for- B C F6 P5 L29 mat. The termini are labeled m-loop with N-ter and C-ter, and the cysteine residues are labeled K21 with their residue numbers. 37 20 180º The n-, m-, and c-loops as K R 16 shown represent the ami- C-ter C15 L C-ter F22 no-terminal, middle, and SCIENCES 13 carboxyl loops, respectively. C35 A

Diagrams were generated APPLIED BIOLOGICAL 11 using MOLMOL. (D)Aspace- C 24 filling model showing the lo- 33 26 F C A E8 cation of different types of C25 amino acids (green, hydro- phobic; blue, positively R30 4 28 charged; and red, negatively n-loop C c-loop P 5 6 charged). (B)and(D)were 29 P F N-ter N-ter L displayed by Weblab View- lite 4.2 and (C) by MOLMOL.

Zhu et al. PNAS ∣ May 29, 2012 ∣ vol. 109 ∣ no. 22 ∣ 8497 Downloaded by guest on September 29, 2021 α αβ Table 1. Lethal concentrations (CL) of micasin on sensitive bacteria following the -helix. Although the presence of the CS has been shown to be a prerequisite for structure and function of de- Microorganism CL (μM) fensins, a strictly defined structure is not often associated with Gram-positive bacteria high antimicrobial activity (18). In micasin, the flexible loop is Bacillus megaterium CGMCC 1.0459 0.054 structurally located in a previously recognized “γ-core” region Bacillus subtilis CGMCC 1.2428 2.03 that is a common functional domain of many AMPs with phylo- Staphylococcus aureus MRSA P1386 2.46 Staphylococcus aureus MRSA P1374 3.35 genetically diverse origins (19). Staphylococcus epidermidis PRSE P1389 11.55 Staphylococcus MRCNS P1369 12.28 Micasin Kills Bacteria with a New Mode of Action. When assayed for Staphylococcus aureus CGMCC1.89 MSSA 17.53 in vitro antimicrobial effect on a broad spectrum of microorgan- þ − Staphylococcus epidermidis PSSE P1111 23.42 isms, micasin showed potent activity against both G and G bac- Micrococcus luteus CGMCC 1.0290 NA teria (Table 1), but had no effect on five filamentous fungi tested Bacillus cereus CGMCC 1.1626 NA or on the yeast Saccharomyces cerevisiae (Table S4). Of all the Gram-negative bacteria sensitive bacteria, micasin displayed the strongest activity against Pseudomonas aeruginosa CCTCC AB 91095 0.94 B. megaterium with a lethal concentration (CL) of 54 nM, and it Agrobacterium tumefaciens CCTCC AB 92026 4.24 ATCC 25922 NA also killed two clinical isolates of MRSA and the opportunistic pathogen P. aeruginosa at low micromolar concentrations. P. aer- NA, no activity, indicating that no inhibition zone was observed uginosa causes significant nosocomial infections, and in most ∕ at 1.0 nmol peptide well. cases it is rather difficult to treat as it has an additional outer membrane permeability barrier (4). The killing kinetics of mica- between residues 7–18 and a β-sheet consisting of two antiparallel sin against B. megaterium showed that it killed this bacterium strands between residues 22–26 and 32–36. A short sequence of more rapidly than vancomycin (Fig. 4A), an inhibiting only three residues (20–22) links the helix to strand I. The loop bacterial cell-wall biosynthesis. The extent of bacterial death connecting the two β-strands (residues 27–31) is highly flexible, caused by micasin and vancomycin is approximately 100% and 4 5 with no dominant turn type defined. The Cys -Pro amide bond 78.5%, respectively, after 3 h of incubation with the antibacterial adopts an unusual cis-conformation, as observed in two Mollusca- agents. The ability of micasin to permeabilize the bacterial mem- derived AITDs (MGD-1 and Cg-DEF). The three disulfide bonds brane was also assessed with the fluorescent nucleic acid-binding are arranged as in a typical CSαβ motif; that is, the Cys4-Cys25 dye propidium iodide. Vancomycin and meucin-18 (a pore-form- disulfide bridge connects the N-terminus to the latter part of ing peptide from scorpion venom) were used as negative and strand I, and the Cys11-Cys33, Cys15-Cys35 disulfide bridges positive control (20, 21). No fluorescence increase was observed connect the helix to strand II. Overall, micasin lacks amphipathic over 10 min after B. megaterium were exposed to micasin and architecture, as identified by its cationic and hydrophobic resi- vancomycin at 5× CL or 10× CL (Fig. 4B), indicating the bacterial dues scattered on the structural surface (Fig. 3D). membrane integrity was not destroyed. On the contrary, meucin- As expected, micasin shares the highest structural similarity to 18 caused an immediate fluorescence increase upon exposure three AITDs (plectasin, MGD-1 and Cg-Def) with a root mean of the peptide. These observations are in agreement with square deviation (rmsd) of 2.73–4.11 Å (Fig. S2B). Micasin and features of these two control molecules previously reported plectasin have different structurally flexible regions, and the func- (20, 21). At a concentration up to 20× CL, micasin only led to tional significance of these regions have been highlighted in other weak dye uptake after 6 min. The lack of membrane permeability defensins (18). In micasin, a highly flexible loop exists between to the dye in the range of lethal concentrations suggests that two rigid β-strands, while in plectasin the variable regions are micasin is not a membrane disruptive AMP, consistent with its located in the first three amino-terminal residues and the loop overall nonamphiphilic architecture (Fig. 3D).

Fig. 4. Effect of micasin on B. megaterium.(A) Killing kinetics of micasin. The cells treated with micasin or vancomycin at 5× CL. The CL value of vancomycin to B. megaterium is 0.119 μM, which is about twofold higher than that of micasin. Medium is without peptide. (B) Effect of micasin on bacterial membrane integrity. In each run, micasin at 5×,10× or 20× CL was added when the ba- sal fluorescence remained con- stant for 200 s. Vancomycin and meucin-18 (CL:0.25μM) were used as negative and positive control, respectively. (C)Scanning and (D) transmission electron mi- croscopic observations of B. megaterium cells in the absence or presence of micasin. The cells were incubated with micasin at 5× CL for 90 min at 37 °C. The particle-like materials are indi- cated by white arrows.

8498 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1201263109 Zhu et al. Downloaded by guest on September 29, 2021 To further explore micasin’s antibacterial mode of action, we bacterial activity, even over 24 h of incubation in mouse serum, employed electron microscopy techniques to visualize any poten- where the activity was comparable with that in H2O (Fig. 5B). In tial damage caused by the peptide on the morphology and cellular contrast, incubation in serum completely inactivated the activity structures of sensitive bacteria. Scanning electron microscopy re- of vancomycin after 24 h. The potential therapeutic efficacy of vealed no obvious morphological change in B. megaterium treated micasin in providing protection against two lethal infections from with micasin at 5× CL after 90 min when compared with the nor- S. aureus MRSA P1386 and P. aeruginosa was evaluated by a mal cells (Fig. 4C), which is different from plectasin that changes mouse peritonitis model. In these assays, all eight mice in the ve- bacterial morphology due to inhibiting cell-wall biosynthesis by di- hicle-treated control group died after 24 h of inoculation of S. rectly binding to its precursor (21). However, treatment with mi- aureus MRSA P1386 or P. aeruginosa, whereas most of the mice casin at this dose led to an effect on the bacterial intracellular treated with micasin survived the 7-day study period with a sur- components as revealed by transmission electron microscopy. vival rate of 79.5 5.7% for S. aureus MRSA P1386 (Fig. 5C) and The micasin-treated cells accumulated some internal protein 91.7 5.7% for P. aeruginosa (Fig. 5D). All the deaths occurred particle-like materials that were not observed with untreated B. before the third day of inoculation. Overall, the protection effi- megaterium cells (Fig. 4D), suggesting that micasin kills bacterial ciency of micasin is comparable with vancomycin. cells via an intracellular action mode to affect protein folding. A similar mode of action was previously proposed for several insect Discussion AMPs whose killing activity is associated with the inhibition of is the first antibiotic isolated from the fungus Penicil- chaperone-assisted protein folding (3). A plant defensin NaD1 lium chrysogenum (23). From 1930 to 1962, more than 20 unique with the CSαβ motif was reported to enter fungal cells and enhance classes of antibiotics were produced and marketed. However, killing via interacting with intracellular targets (22). The detailed since then the world has made only two new classes of antibiotics mechanism of action of micasin awaits further investigation. (24, 25). The discovery of peptide antibiotics in diverse fungal species is thus of considerable importance, as it demonstrates that Micasin Is a New Antiinfective Peptide with Therapeutic Potential. fungi are a rich resource of human medicines to be exploited. The Micasin caused no notable hemolysis for mammalian erythro- protection from infections of S. aureus and P. aeruginosa by mi- cytes at concentrations up to 50 μM (Fig. 5A). Moreover, it casin indicates that relative to mammalian AMPs (7, 26), fungal exhibited high serum stability as identified by no decrease of anti- DLPs have more advantages as human drugs owing to their SCIENCES APPLIED BIOLOGICAL

Fig. 5. Evaluation of therapeutic potential of micasin. (A) Hemolytic effect of micasin on mouse red blood cells. Meucin-49, a scorpion venom-derived cytolytic peptide of 49 residues, was used as a positive control (this peptide will be published elsewhere). Cells were incubated with different concentrations of peptides for 30 min at 37 °C. Controls for 0 and 100% heamolysis were determined by PBS buffer and 1% Triton X-100, respectively. Percentage haemolysis (%) are expressed as mean SD (n ¼ 3). (B) Serum stability of micasin. The peptide was incubated at 37 °C in fresh normal mouse serum for 0, 1, 3, 6, and 24 h. Residual activities were measured by inhibition-zone assays with B. megaterium.(C)and(D) Survival percentage of mice after peritoneal infection with S. aureus MRSA P1386 (C) and P. aeruginosa (D). Treatment with micasin or vancomycin was initiated 1 h after inoculation. In these two models, 0.9% NaCl was used in the vehicle group whose survival fraction was 0 out of 8 mice (P ≤ 0.0001).

Zhu et al. PNAS ∣ May 29, 2012 ∣ vol. 109 ∣ no. 22 ∣ 8499 Downloaded by guest on September 29, 2021 potent therapeutic efficacy associated with the high antibiotic ac- johnsonii, and P. aeruginosa) frequently inhabiting human skin, tivity and serum stability as well as low toxicity. which provide protection from other microbial infections (28). In the initial stage of this study, we attempted to isolate micasin One such example is that P. aeruginosa in human skin produces as well as micasin-1 from the liquid culture of M. canis by tradi- compounds to kill and inhibit fungal growth (28). In addition, S. tional biochemical techniques, but failed. This result was prob- epidermidis was also found to generate phenol-soluble antimicro- ably due to their low abundance in the culture because their bial agents to contribute to normal defense at the epidermal in- relatively rare trascripts can be only amplified by two rounds terface (29). In this case, we assume that as common pathogens of of nested PCR. In fact, plectasin was also identified by a genetic animal and human skin (30), dermatophytes firstly need to fight approach, instead of biochemical purification, to trap cDNA against the bacterial flora by producing defensins to successfully clones encoding a signal peptide by a transposon-assisted signal survive on human skin. Consistent with this assumption, among trapping system (10). However, such a genetic approach depends the skin bacteria, three (S. epidermidis, S. aureus and P. aerugino- on large-scale random sequencing. Alternatively, the resource of sa) were used in this study and all are sensitive to micasin; in sequenced fungal genomes will accelerate our study in that it can contrast, it had no effect on M. canis itself and the gut-derived rapidly recognize DLPs in a given fungal species, and then a series Escherichia coli. This finding supports a potential role of micasin of molecular and biochemical experiments as well as functional in competition with bacterial flora and suggests that dermatophy- assays can be performed. We found that nearly all the DLPs iden- tic defensins (i.e., micasin, tritosin, trivesin and trirusin) could be tified here are derived from Pezizomycotina (Ascomycota). Such particularly useful in developing drugs against human skin- a species-specific phylogenetic distribution should not be due to derived opportunistic pathogens. sampling bias, because there are only six species of Pezizomyco- tina whose genomes were completely sequenced; in Saccharomy- Materials and Methods cotina (Ascomycota), the number is thirteen. It is known that The database search strategy for finding new defensin-like genes has been Ascomycota is the largest phylum of Fungi, comprising almost described previously (12). Briefly, amino acid sequences of plectasin and re- 50% of all known fungal species, and Pezizomycotina is the lar- lated defensins were first used as a query to perform TBLASTN search of fun- gest subphylum of Ascomycota, containing more than 32,000 de- gal genomic database (http://www.ncbi.nlm.nih.gov) under the default scribed species (27), which constitute a tremendous resource parameters. This database includes 134 species of fungi with 26 genomes completely sequenced. The presence of a signal peptide combined with a library of templates for molecular design. For example, current “hallmark” defensin cysteine motif was used to filter the hits. fungal geneome projects include at least 74 Pezizomycotina spe- Additional experimental procedures are provided in SI Materials and cies whose genomes are being sequenced. Methods As Pezizomycotina species occur in numerous ecological niches and occupy virtually all terrestrial and aquatic ecosystems, ACKNOWLEDGMENTS. We thank the Lab of Bio-imagine, the Institute of it remains an open question as to why Pezizomycotina rather than Biophysics for our electron microscopy work, and we are grateful to Lei other subphylums of Ascomycota retains DLPs during evolution. Sun for her help in making TEM samples and taking TEM images. We also The antibacterial spectrum of micasin could provide clues to thank Dr. Dongming Li (Beijing University, China) for kindly providing M. canis. This work was supported by the National Natural Science Founda- answer this question. Molecular detection has identified seven tion of China (Grant 30730015 and 30921006) and the National Basic bacterial species (S. epidermidis, S. aureus, Streptococcus mitis, Research Program of China (Grant 2010CB945300). D.J.C. is a National Health Propionibacterium acnes, Corynebacterium spp., Acinetobacter and Medical Research Council (Australia) Professorial Fellow.

1. Zasloff M (2002) of multicellular organisms. Nature 16. Laskowski RA, Rullmannn JA, MacArthur MW, Kaptein R, Thornton JM (1996) AQUA 415:389–395. and PROCHECK-NMR: programs for checking the quality of protein structures solved 2. Brogden KA (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in by NMR. J Biomol NMR 8:477–486. – bacteria? Nat Rev Microbiol 3:238–350. 17. Hutchinson EG, Thornton JM (1996) PROMOTIF a program to identify and analyze – 3. Hale JD, Hancock RE (2007) Alternative mechanisms of action of cationic antimicrobial structural motifs in proteins. Protein Sci 5:212 220. peptides on bacteria. Expert Rev Anti Infect Ther 5:951–959. 18. Landon C, Barbault F, Legrain M, Guenneugues M, Vovelle F (2008) Rational design of peptides active against the gram positive bacteria Staphylococcus aureus. Proteins 4. Srinivas N, et al. (2010) Peptidomimetic antibiotics target outer-membrane biogenesis 72:229–239. in Pseudomonas aeruginosa. Science 327:1010–1013. 19. Yeaman MR, Yount NY (2007) Unifying themes in host defence effector polypeptides. 5. Easton DM, Nijnik A, Mayer ML, Hancock RE (2009) Potential of immunomodulatory Nat Rev Microbiol 5:727–740. host defense peptides as novel anti-infectives. Trends Biotechnol 27:582–590. 20. Gao B, Sherman P, Luo L, Bowie J, Zhu S (2009) Structural and functional characteriza- 6. Peschel A, Sahl HG (2006) The co-evolution of host cationic antimicrobial peptides and tion of two genetically related meucin peptides highlights evolutionary divergence – microbial resistance. Nat Rev Microbiol 4:529 536. and convergence in antimicrobial peptides. FASEB J 23:1230–1245. 7. Vaara M (2009) New approaches in peptide antibiotics. Curr Opin Pharmacol 21. Schneider T, et al. (2010) Plectasin, a fungal defensin, targets the bacterial cell wall 9:571–576. precursor Lipid II. Science 328:1168–1172. 8. Marr AK, Gooderham WJ, Hancock RE (2006) Antibacterial peptides for therapeutic 22. van der Weerden NL, Hancock RE, Anderson MA (2010) Permeabilization of fungal use: obstacles and realistic outlook. Curr Opin Pharmacol 6:468–472. hyphae by the plant defensin NaD1 occurs through a cell wall-dependent process. 9. Eckert R (2011) Road to clinical efficacy: challenges and novel strategies for antimicro- J Biol Chem 285:37513–37520. bial peptide development. Future Microbiol 6:635–651. 23. Fleming A (1929) On the antibacterial action of cultures of a Penicillium, with special 10. Mygind PH, et al. (2005) Plectasin is a peptide antibiotic with therapeutic potential reference to their use in the isolation of B. influenza. Br J Exp Pathol 10:226–236. from a saprophytic fungus. Nature 437:975–980. 24. Coates A, Hu Y, Bax R, Page C (2002) The future challenges facing the development of – 11. Hara S, et al. (2008) Plectasin has antibacterial activity and no affect on cell viability or new antimicrobial drugs. Nat Rev Drug Discov 1:895 910. IL-8 production. Biochem Biophys Res Commun 374:709–713. 25. Coates A, Halls G, Hu Y (2011) Novel classes of antibiotics or more of the same? Br J Pharmacol 163:184–194. 12. Zhu S (2008) Discovery of six families of fungal defensin-like peptides provides insights 26. Hancock RE, Sahl HG (2006) Antimicrobial and host-defense peptides as new anti- into origin and evolution of the CSαβ defensins. Mol Immunol 45:828–838. infective therapeutic strategies. Nat Biotechnol 24:1551–1557. 13. Wang Y, Zhu S (2011) The defensin gene family expansion in the tick Ixodes scapularis. 27. Kirk PM, Cannon PF, David JC, Stalpers JA (2010) Ainsworth and Bisby’s dictionary of – Dev Comp Immunol 35:1128 1134. the Fungi (CAB International, Wallingford, UK), 9th Ed. 14. Gao B, Zhu L, Zhu S (2011) A naturally-occurring carboxyl-terminally truncated 28. Cogen AL, Nizet V, Gallo RL (2008) Skin microbiota: a source of disease or defence? alpha-scorpion toxin is a blocker of sodium channels. Biochem Biophys Res Commun Br J Dermatol 158:442–455. 411:673–678. 29. Cogen AL, et al. (2010) Selective antimicrobial action is provided by phenol-soluble 1 13 15 15. Wishart DS, Bigam CG, Holm A, Hodges RS, Sykes BD (1995) H, C and N random modulins derived from Staphylococcus epidermidis, a normal resident of the skin. coil NMR chemical shifts of the common amino acids. I. Investigations of nearest- J Invest Dermatol 130:192–200. neighbor effects. J Biomol NMR 5:67–81. 30. Hainer BL (2003) Dermatophyte infections. Am Fam Physician 67:101–108.

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