Mycologia. 91 (2), 1999, pp. 269-277. © 1999 by The Mycological Society of America, Lawrence, KS 66044-8897 Penicillium pimiteouiense: a new species isolated from polycystic kidney cell cultures Stephen W. Peterson 1,2 (PKD) , fungi appeared in some cell culture bottles Microbial Properties Research Unit, National Center (Miller-Hjelle et al 1997). Contemporaneous epithe­ for Agricultural Utilization Research, Agricultural lial cell cultures originating with nondiseased kidney Research Service, Us. Department ofAgriculture, cells did not display fungal growths. Initial examina­ 1815 N. University St., Peoria, Illinois 61604 tion of the fungus on the basis of phenotypic data Sylvia Corneli (Raper and Thorn 1949, Pitt 1979, Ramirez 1982) Mycotoxin Research Unit, National Center for showed that the fungus resembled several monover­ Agricultural Utilization Research, Agricultural Research Service, us. Department ofAgriculture, ticillate species, Penicillium restrictum Gilman & Ab­ 1815 N. University St., Peoria, Illinois 61604 bot, P. dimorphosporum Swart, and P. striatisporum ]. Thomas Hjelle Stolk. Detailed phenotypic examination showed that Marcia A. Miller-Hjelle this strain did not fit the description of any of these Deborah M. owak species. Department of Biomedical and Therapeutic Sciences, Lobuglio et al (1994) and Peterson (1998) have University ofIllinois School ofMedicine at Peoria, One shown that ribosomal DNA (rD A) sequences can be Illini Drive, Peoria, Illinois 61605 used to distinguish the species of Penicillium and to Paul A. Bonneau determine their phylogenetic relationships. Ribosom­ Microbial Properties Research Unit, National Center al DA from each of these species was amplified us­ for Agricultural Utilization Research, Agricultural ing PCR, sequenced, and analyzed by maximum par­ Research Service, us. Department ofAgriculture, simony. The strains from PKD cell cultures had rD A 1815 N. University St., Peoria, Illinois 61604 sequences that were distinct from the three species it resembles and from all other known Penicillium Abstract: As part of a study of polycystic kidney dis­ species (Peterson 1998). The monoverticillate peni­ ease (PKD), epithelial cells were isolated and propa­ cillus of the new species would place it in subgenus gated from cyst walls of human kidneys obtained at A spergilloides, but Peterson (1998) has shown that nephrectomy. A Penicillium species was found grow­ subgenus Aspergilloides is not monophyletic. Its phy­ ing with the epithelial cells in some culture flasks. It logenetic relationship to other species was examined was monoverticillate and somewhat resembled P. res­ by parsimony analysis with species from subgenera trictum but differed morphologically from this and all Aspergillioides and Furcatum (Pitt 1979). other Penicillium species. Two strains of this fungus Ochratoxin A is known to cause nephritis of kid­ were isolated in pure culture and described as the neys when ingested (Creppy 1995). Although ochra­ new species Penicillium pimiteouiense. Ochratoxin A toxin A is known from only four species of Aspergillus production was not detected in P. pimiteouiense. The and Penicillium, and although ochratoxin A may not role, if any, that this fungus plays in polycystic kidney be produced by the fungus growing in tissue, we as­ disease is not known. sayed the new species for production of ochratoxin Key Words: fungi, polycystic kidney disease, A because of its habitat. rD A, systematics, Trichocomaceae MATERIALS D METHODS I TTRODUCTIO Media and cultures.-Czapek's agar (Cz) (Raper and Thorn 1949), Czapek's yeast extract agar (CYA) , Blakeslee's extract In the course of growing kidney epithelial cell lines MEA, and G25 agar (Pitt 1979) were formulated as de­ from kidneys displaying polycystic kidney disease scribed. Czapek's agar with 20% sucrose (Cz20) and M40Y agar were made as described (Raper and Fennell 1965). Accepted for publication October 15, 1998. Colony transfers were made by touching a sterile inoculat­ I E-mail: [email protected] ing wire to a sporulating colony, dispersing the conidia in 2 Tames are necessary to report factually on available data; how­ ca 50 jJ..L of sterile 0.5% Kodak photoflo, dipping the in­ ever, the SDA neither guarantees nor warrants the standard of the product, and the use of the name by SDA implies no approval oculating wire in the conidial suspension and touching it of the product to the exclusion of others that may also be suitable. to the agar surface. For identification using the keys of Pitt 269 270 MYCOLOGIA (1979), the cultures were grown on CYA at 5 C, 25 C, and method of Breitholtz et al (1991) on plasma with some mod­ 37 C; on G25 at 25 C; and on MEA at 25 C for 7 d in the ifications: 2 mL of sample were acidified with 500 ILL of HCI dark. For identification using the keys of Raper and Thorn concentrate solution, extracted with 10 mL of methylene (1949) and Ramirez (1982), the cultures were grown on Cz chloride and centrifuged at 2000 g for 10 min. The bottom and MEA for 7-10 d at 25 C in the dark. Cultures were also layer was collected and evaporated to dryness under a nitro­ grown on Cz20 and M40Y for 7 d at 25 C to assess growth gen stream, reconstituted with 1 mL of acetonitrile/water on media with reduced water activity. (50:50) solution, filtered with a 0.2 ILm, 13 mm syringe filter (Whatman Inc., Clifton, New Jersey) and 50 ILL portions of Origins offungal growths.-Fungal growth was observed in sample were subjected to HPLC analyses. The HPLC column kidney cell cultures obtained from two separate kidneys. used was a RP-18 Spheri-5, 5 ILm, 220 X 4.6 mm from Perkin The kidneys were obtained from patients with autosomal Elmer Applied Biosystems Div., Foster City, California (ABI) , dominant polycystic kidney disease: Patient #1, a 43-year-old maintained at 30 C during the analyses. The mobile phase female, had undergone peritoneal dialysis for two yr prior consisted of acetonitrile/water/acetic acid (50:49:1) with a to nephrectomy; Patient #2, a 47-year-old female, had un­ flow rate of 1.0 mL/min, and the fluorometric detector dergone hemodialysis continuously for nearly 10 yr prior to (Spectra System FL2000, Spectra-Physics, San Jose, Califor­ nephrectomy. The kidneys were obtained 17 mo apart. Cells nia) was set at 333 nm excitation and 470 nm emission. The lining the inner (urinary) surface of kidney cysts were iso­ average recovery value for sample spiked with 10 ILg/Kg OA lated from each kidney by digestion with warm collagenase in Delbecco's Modified Essential Medium (DMEM) nutri­ was 96% (standard deviation: 2.4). ent mixture F-12 HAM (Sigma Chemical Co., St. Louis, Mis­ DNA extraction and sequencing.-Five mm plugs of agar and souri) (Hjelle et al 1990). The cells obtained were pelleted, mycelium were cut from a MEA plate and placed in a 15­ the collagenase solution discarded, the cells resuspended in mL sterile disposable centrifuge tube. Two mL of DA ex­ DMEM/F12 HAM, and fortified with 10 mg/mL strepto­ traction buffer (100 mM Tris, 50 mM EDTA, 1% sarcosyl, mycin, 2 mM glutamine, 0.25 ug/mL amphotericin B, 3 g/ pH 8.0), 2 g glass beads (0.5 mm diam) and 2 mL of phe­ L sodium bicarbonate, 10% bovine calf serum (Hyclone nol : chloroform (1: 1) were added. Cell walls were disrupted Lab Inc., Logan, Utah), and seeded in plastic T-25 flasks for and an emulsion was formed by vortexing the tube for 45­ growth at 37 C in humidified 95% air/5% CO , All culture 2 60 s. Proteins were extracted by gentle rocking of the emul­ solutions used in cell isolation and propagation were devoid sion for 20 min. Organic and aqueous phases were separat­ of microbial growth for two mo, the endpoint for the ste­ rility checks. ed by low speed centrifugation (ca 2000 g) for 5 min. The Single colonies of the Penicillium sp. were picked from aqueous phase was pipetted to a clean tube, 0.1 vol 3M kidney cell culture bottles with a sterile inoculating wire and sodium acetate and 1.3 vol 95% ethanol were added to pre­ transferred to MEA petri plates. After 4-5 d growth, conidia cipitate D A. The precipitate was pelleted by low speed were picked from this plate, dispersed in dilute sterile pho­ centrifugation (ca 2000 g), and the liquid was decanted. toflo and ca 50 colonies were spotted on MEA plates from The pellet was dissolved in 100 ILL of TE (10 mM Tris, 1 the conidial suspension. Colonies were checked daily for the mM EDTA, pH 8.0), 2.5 vol of saturated NaI was added, appearance of mixed cultures. After 10 d, one of the pure and DA was adsorbed to a silica matrix (Geneclean, colonies was subcultured and kept for further study. Bio101, La Jolla, California) following the manufacturer's All strains and species of Penicillium used in this study are instructions. The matrix was rinsed to remove impurities. permanently preserved in the Agricultural Research Service Genomic DA was eluted into 1/10th strength TE buffer Culture Collection ( RRL), Peoria, Illinois, as lyophilized and stored at - 20 C. cultures. The strains, their origins, and GenBank deposit A fragment of the ribosomal DA repeat unit that in­ numbers for the nucleotide sequences are listed in TABLE 1. cludes ITS1, ITS2, 5.8S rD A and about 635 bases of the 28S rD A (regions D1 and D2) was amplified using PCR SEM microscopy.-Blocks of agar with mycelium were cut and site-specific oligonucleotide primers (Peterson et al from MEA plates and placed in screw-eap vials with 0.5 mL 1998, White et al 1990). The PCR amplified fragment was of 1% OS04 in cacodylate buffer (Peterson 1992).