CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2057 - 1/11
`
`

`
`© 2007 Elsevier Masson SAS. All rights reserved.
`Elsevier Masson SAS, au capital de 675 376 €
`- 542 037 031 RCS Nanterre
`
`This journal and the individual contributions contained in it are protected under copyright by Elsevier Masson SAS, and the
`following terms and conditions apply to their use:
`
`Photocopying
`
`Single photocopies of single articles may be made for personal use as allowed by national copyright laws. Permission of the
`Publisher and payment of a fee is required for all other photocopying, including multiple or systematic copying, copying for
`advertising or promotional purposes, resale, and all forms of document delivery. Special rates are available for educational
`institutions that wish to make photocopies for non—profit educational classroom use.
`Permissions may be sought directly from Elsevier’s Rights Department in Oxford, UK: phone (+44) 1865 843830,
`fax (+44) 1865 853333, e-mail permissions@elsevier.com. Requests may also be completed on—line via the Elsevier homepage
`(littp://www.elsevier.com/locate/permissions).
`In the USA, users may clear permissions and make payments through the Copyright Clearance Center, Inc., 222 Rosewood Drive,
`Danvers, MA 01923, USA; phone: (+1) (978) 7508400, fax: (+1) (978) 7504744, and in the UK through the Copyright Licensing
`Agency Rapid Clearance Service (CLARCS), 90 Tottenham Court Road, London WIP OLP, UK; phone: (+44) 20 7631 5555;
`fax: (+44) 20 7631 5500. Other countries may have a local reprographic rights agency for payments.
`
`Derivative Works
`
`Subscribers may reproduce tables of contents or prepare lists of articles including abstracts for internal circulation within their
`institutions. Permission of the Publisher is required for resale or distribution outside the institution. Permission of the Publisher
`is required for all other derivative works, including compilations and translations.
`
`Electronic Storage or Usage
`
`Permission of the Publisher is required to store or use electronically any material contained in this journal, including any article or
`part of an article. Except as outlined above, no part of this publication may be reproduced, stored in a retrieval system or transmitted
`in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without prior written permission of the
`Publisher. Address permissions requests to: Elsevier Rights Department, at the fax and e-mail addresses noted above.
`
`Notice
`
`No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability,
`negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material
`herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages
`should be made. Although all advertising material is expected to confonn to ethical (medical) standards, inclusion in this publication
`does not constitute a guarantee or endorsement of the quality or value of such product or of the claims made of it by its manufacturer.
`
`Advertising information. Advertising orders and enquiries can be sent to: Katrina Barton, Print Operations Co—ordinator,
`Elsevier, 84 Theobalds Road, London WClX 8RR, UK; phone +44 (0) 20 7611 4117; e-mail: k.barton@elsevier.com
`
`English language help service: Upon request, Elsevier will direct authors to an agent who can check and improve the English of
`their paper (bejore siibinission). Please contact authorsupport@elsevier.com for further information.
`
` For full and complete Instructions to Authors, please refer to http://authors.e1sevier.com/journal/micinf
`
`Available online at
`" ~’~’ at?’
`
`ScienceDirect
`
`www.sciencedirect.com
`
`if
`
`Microbes and Infection has no page charges
`_J
`CFAD V. Anacor, |PR20‘l5-01776 ANACOR EX. 2057 - 2/11
`69 The paper used in this publication meets the requirements of ANSI/NISO Z39.48—1992 (Permanence of Paper)
`
`
`
`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2057 - 2/11
`
`

`
`
`
`Vol. 9, issue 12-13, October 2007
`
`CONTENTS
`ELSEVIE
`www.e1sevier.com/locate/micinf
`
`
`
`1
`
`Cited in: Medline, Excerpta Medica/Embase, Current Contents (Life Sciences), Science Citation Index
`
`
`Original articles
`
`G1‘—1+ cells play an essential role in an experimental model of disseminated histoplasmosis
`A. Sci-Nzmes, A.I. Medeiros, C.A. Sorgi, E.G. Soares, C.M.L. Mafiei, C.L. Silva and L.H. Faccioli
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`1393
`
`The bacterial metabolite 2,3—butanediol ameliorates endotoxin—induced acute lung injury in rats
`S.-C. Hsieh, C.—C. Lu, Y.-T. Horng, P.—C. Soo, 1’.-L. Chang,
`l’.—H. Tsai, C.-S. Lin and H.—C. Lai .
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`1402
`
`Chlamydophilal antigens induce foam cell formation via c—Jun NH2—termina1 kinase
`T. Kirazawa, A. Fukushima, S. 0/aigawa, S. Yanagimoto, K. Tsukada, K. Tatsuno, K. Koike, S. Kimura,
`T Kishimoto,
`1’. Shibasaki and 1’. Ota .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`1410
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`1415
`
`CDNA representational difference analysis used in the identification of genes expressed by Trichophyron
`rubrum during Contact with keratin
`L.C. Baeza, A.M. Bailcio, C.L. Borges, M. Pereira, C.M. de A. Soares and M.J.S. Mendes Giannini
`
`.
`
`'
`*
`
`Visualization of microtubule-mediated transport of influenza viral progeny ribonucleoprotein
`F Momose, 1/. Kikachi, K. Komase and 1/. Morikawa .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`Identification of proteins directly phosphorylated by UL13 protein kinase from herpes simplex virus 1
`R. Asai, T. Ohno, A. Kato and 1’. Kawaguchi .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`1422
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`1434
`
`Extended immunization intervals enhance the immunogenicity and protective efficacy of plasmid DNA vaccines
`G.’1". Brice, C. Dobafio, M. Sedegah, M. Stefaniak, N.L. Graber, J.J. Campo, D.J. Carucci anal D.L. Doolan .
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`1439
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`1447
`
`Preexisting anti-Salmonella vector immunity prevents the development of protective antigen—specific
`CD8 T-cell frequencies against murine listeriosis
`V.E. Sevil Doménech, K. Panthel, K.M. Meinel, S.E. Winter and H. Riissmann .
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`i
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`Antibody response against saliva antigens of Anopheles gambiae and Aedes aegypli in travellers in tropical Africa
`E. 0rlandi—Pradines, L. Almeras, L. Denis dc Senneville, S. Barbe, F. Remoué, C. Villard, S. Cornelie,
`K. Pen/mat, A. Pascual, C. Bourgouin, D. Fontenille, J. Bonner, N. Corre-Catelin, P. Rcite); F. Pagés,
`D. Laffite, D. Boulanger, F. Simondon, B. Pradines, T. Fusai anal C. Rogier .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`1454
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`1463
`
`CD44, a signal receptor for the inhibition of the cytoadhesion of CD36—binding Plasmodium._falciparLm1—infected
`erythrocytes by CSA—binding infected erythrocytes
`C. Jnrzynski, J. Gysin and B. Pouvelle .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`The role of human innate immune factors in nasal colonization by Staphylococcus aureus
`A. van Bel/cum, M. Emonrs, H. Wertheim, C. ale Jongh, J. Nonwen, H. Barrels, A. Cole, A. Cole, P. Hermans,
`H. Boelens, N.L.-d. Toom, S. Snijclers, H. Verbrugh and W. van Leeuwen .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`1471
`
`Systemic but not mucosal immunity induced by AVA prevents inhalational anthrax
`D.M. Klinman, D. Currie, G. Lee, 1/. Grippe ana' T. Merkcl
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`1478
`
`A surface 75-kDa protein with acid phosphatase activity recognized by monoclonal antibodies that inhibit
`Paracocciclioides brasiliensis growth
`P. Xander, AF Vigna, L. dos S. Feitosa, L. Pugliese, A.M. Bailrio, C.M. de A. Soares, R.A. Morfara,
`M. Mariano and J.D. Lopes .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`1484
`
`
`
`CFAD V. Anacor, |PR2015-01776 ANACOR E)(_. 057 - 3/11
`
`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2057 - 3/11
`
`

`
`CONTENTS (continued)
`
`In vitro cultured peripheral blood mononuclear cells from patients with chronic schistosomiasis mansoni
`show immunomodulation of cyclin D1 ._, 3 in the presence of soluble egg antigens
`A.C. Campi-Azevedo, G. Gazzinelli, M.E. Bottazzi, A. Teixeira-Carvalho, R. Corréa-Oliveira and 1.R. Caldas .
`
`.
`
`.
`
`Arginine-specific gingipain A from Porphyromonas gingivalis induces Weibel—Palade body exocytosis
`and enhanced activation of vascular endothelial cells through protease-activated receptors
`M. lnomata, T Into, Y. Is/u'/zara, M. Nakcls/1ima, T. Noguchi and K. Matsushita .
`.
`.
`.
`.
`.
`.
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`1493
`
`1500
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`Short communication
`
`Selective transport of staphylococcal enterotoxin A through in vitro generated human M cells
`M. Maresca, E. Dumay, J. Fcmtini and B. Caporiccio .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`1507
`
`
`
`CFAD V. Anacor, |PR2015-01776 ANACOR EX. 2057 - 4/11
`
`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2057 - 4/11
`
`

`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`
`LSEVII-zii
`
`E’?
`INSTITUT PASTEUR
`
`Microbes and Infection 9 (2007) 1415-1421
`
`Original article
`
`
`
`www.elsevier.com/locate/inicinf
`
`CDNA representational difference analysis used in the
`identification of genes expressed by Tric//zophyron rubrum
`during Contact with keratin
`
`Lilian Cristiane Baeza “, Alexandre Melo Bailao b, Clayton Luiz Borges b, Maristela Pereira 1”,
`Célia Maria de Almeida Soares b, Maria Jose Soares Mendes Giannini “”“
`
`" Laborurério do Mica/ogia CIfnica, Doparlamcnro do /lncilixes ClfI1i('as, Fucu/dude dc Ciéncias Farmacéuricas,
`UNESP, CEP 14801-902, 162/ AI‘aI‘a(/Ham, SP, Brazil
`1’ Laborut(5r1'0 do Ifiologirz Molecular, IllSIfIllf(I dc CiéncI'ur Biolrigiczzs, Universiclzlz/0 Faloral dc Goizis,
`CEP 74001-970, Goicinia, GO, Brazil
`
`Received 10 November 2006; accepted 12 July 2007
`Available online 17 July 2007
`
`Abstract
`
`Dermatophytes are adapted to infect skin, hair and nails by their ability to utilize keratin as a nutrient source. Tric/zap/zyton rubrmn is an
`anthropophilic fungus, causing up to 90% of chronic cases of dermatophytosis. The understanding of the complex interactions between the fun-
`gus and its host should include the identification of genes expressed during infection. To identify the genes involved in the infection process,
`representational difference analysis (RDA) was applied to two CDNA populations from T. rubrum, one transcribed from the RNA of fungus
`cultured in the presence of keratin and the other from RNA generated during fungal growth in minimal medium. The analysis identified differ-
`entially expressed transcripts. Genes related to signal transduction, membrane protein, oxidative stress response, and some putative virulence
`factors were up-regulated during the contact of the fungus with keratin. The expression patterns of these genes were also verified by real-
`time PCR, in conidia of T. rubrum infecting primarily cultured human keratinocytes in virro, revealing their potential role in the infective pro-
`cess. A better understanding of this interaction will contribute significantly to our knowledge of the process of dermatophyte infection.
`© 2007 Elsevier Masson SAS. All rights reserved.
`
`Keywo/'d.r.' Triclzoplzyton rubrum; Representational difference analysis; Infection; Dermatliophytoses
`__
`
`1. Introduction
`
`Dermatophytoscs are among the few fungal diseases that
`are directly communicable from person to person. Dermato-
`phytes infect mainly healthy individuals, causing infections
`of keratinized structures,
`including the skin, hair, and nails
`[1]. Dermatophytes are not part of the normal human 1nicro—
`bial flora. They are, however, particularly well adapted to in-
`fecting these tissues because, unlike most other microbial
`pathogens, they can use keratin
`a source of nutrients [2].
`
`Corresponding author. Tel.: +55 (16) 3301 6556; fax: +55 (16) 3301 6547.
`E-mail cI(lcl1'e.\‘s.' giannini@fcfar.unesp.br (M.J.S. Mendes Giannini).
`
`Tric/top/'zyz‘0n rubrum is the most frequently isolated agent of
`dermatophytosis worldwide, accounting for approximately
`80% of reported cases of onychomycosis [3]. Since 90% of
`the chronic dermatophyte infections are caused mainly by
`T.
`rubrum,
`this pathogen must have evolved mechanisms
`that evade or suppress cell-mediated immunity [4].
`Despite its prevalence, little is known about the molecular
`basis of dermatophyte pathogenesis. Studies regarding the
`structure, expression, and regulation of the genes of T. rub/"um
`have been relatively limited because of its unaggressive and
`non-life—threatening nature.
`In host—pathogen interactions,
`the gene expression of the pathogen is modulated by signals
`from the host, and knowing the pattern of expression may
`
`1286-4579/EB - see front matter © 2007 Elsevier Masson SAS. All rights reserved.
`doi: l (l. l 0 l 6/j.micinf.2007.07.005
`
`
`
`CFAD V. Anacor, |PR2015-01776 ANACOR EX. 2057 - 5/11
`
`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2057 - 5/11
`
`

`
`1416
`
`L.C. Baeza at al. / 1W1(‘/‘()1)L’S and Infection 9 (2007) 1415-1421
`
`
`
`provide insights into the disease mechanisms [5]. Few poten-
`tial T. rubrimz virulence factors have been examined in detail,
`and most of them are keratinolytic proteases.
`The understanding of the complex interactions between
`fungus and host must
`include the identification of genes
`expressed during infection. An efficient approach to the iden-
`tification of differentially expressed genes in T. rubrum in-
`volves rapid series of subtractive hybridizations of CDNA
`prepared from two cell populations. Representational differ-
`ence analysis (RDA) is a powerful and sensitive tool for the
`identification of differentially expressed genes and enables
`the isolation of both up- and down—regulated genes expressed
`in two different cDNA populations [6]. Recently, this strategy
`was applied to the differentially expressed genes of the human
`pathogenic fungus Paracoccidioides brasiliensis during the
`host interaction, revealing a set of candidate genes that 1’. bra-
`siliensis may express to adapt to the host conditions [7].
`The aim of the present study was to identify genes differen-
`tially expressed in T. rubrum, cultured in the presence and
`absence of keratin to simulate the host infection. The role of
`these genes was corroborated by confirming their induction
`during the infective process in a primary keratinocyte cell
`culture. Our studies provide the first view of the T. rubrum
`transcriptional response to host—-pathogen interaction.
`
`2. Materials and methods
`
`2.1. Strain and culture conditions
`
`T. rubrzmz isolate ATCC 52021 (American Type Culture
`Collection) was cultured for 10 days at 25-28 °C in Sabour-
`aud’s liquid medium and transferred to two different culture
`media:
`(i) a culture referred to as “tester” in liquid Cove’s
`medium [8] supplemented with keratin (Sigma) 100 ug/mL, and
`(ii) a culture named “driver” in Cove’s minimal medium, both
`cultivated for 24 h at room temperature. As a control, a reverse ex-
`periment was conducted in which the driver RNA was extracted
`from keratin culture and the tester RNA from minimal medium.
`
`2.2. RNA isolation and cDNA synthesis
`
`Total RNA was extracted from T. rubrum cultured under
`
`each experimental condition by using the Trizol reagent (Invi-
`trogen Life Technologies, Carlsbad, CA). First strand cDNA
`synthesis was performed with reverse transcriptase (RT Super-
`script III, Invitrogen, Life Technologies) using 1 ug of total
`RNA. The first strand of cDNA was used as template to syn-
`thesize the second strand, by using the SMART PCR cDNA
`synthesis kit (Clontech Laboratories, Palo Alto, CA, USA).
`
`cDNA from RNA extracted from fungus cultured in the pres-
`ence of keratin. The resulting products were purified using
`a GFX kit (GE Healthcare, Chalfont St. Giles, UK). The
`digested tester cDNA was ligated to adapters (a 24—mer
`annealed to a 12—mer). To generate the differential products,
`tester and driver cDNAs were mixed, hybridized at 67 °C for
`18 h and amplified by PCR with the 24—mer oligonucleotide
`primer. Two successive rounds of subtraction and PCR ampli-
`fication were performed with hybridization tester—driver
`ratios of l:l0 and 1:100, respectively. Adapters were changed
`between these cross—hybridizations, and different products
`were purified using the GFX kit [9,l0].
`After the second subtractive reaction, the finally amplified
`CDNA pools were cloned directly into the pGEM-T Easy vec-
`tor (Promega, Madison, USA). Escherichia coli XL] Blue
`competent cells were transformed with the ligation products.
`Selected colonies were picked and grown in deep-well plates.
`Plasmid DNA was prepared from clones using standard proto-
`cols. In order to generate the EST (expressed sequence tags)
`sequences, single-pass, 5’—end sequencing of cDNAs by stan-
`dard fluorescence labeling dye—terminator protocols with T7
`flanking vector primer was peiformed. Samples were loaded
`onto a MegaBACE 1000 DNA sequencer (GE Healthcare)
`for automated sequence analysis.
`
`2.4. EST processing pipeline and differential
`expression analysis
`
`EST sequences were pre-processed using the Phred and
`Crossmatch (http://www.genome.washington.edu/UWGC/ana-
`lysistools/Swat.cfm) programs. Sequences with at least 100
`nucleotides and Phred quality greater than or equal
`to 20
`were considered for further analysis. ESTs were screened for
`vector sequences against
`the UniVec data, and assembled
`with the CAP3 program [11]. The filtered sequences were
`compared against the GenBank (http://www.ncbi.nlm.nih.gov)
`non—redundant (nr) database from the National Center for Bio-
`technology Information (NCBI) using the BLASTX program
`[12], Cluster of Orthologous Groups (COG) and Gene Ontol-
`ogy (GO). MIPS (http://mips.gsf.de/) and InterPro databases
`of protein families, domains and functional sites were used
`to assign functional categories.
`
`2.5. Assay of T. rubrum—-keratinocytes interaction
`
`2.3. Subtractive hybridization and generation of
`subtracted libraries
`
`The CDNA fragments were digested with Sau3AI restric-
`tion enzyme (Promega, Madison, USA). A subtracted cDNA
`library was constructed using driver cDNA synthesized from
`RNA of T. rubrimz cultured in minimal medium and tester
`
`Cultures of keratinocytes were isolated from human breast
`skin obtained from routine plastic surgery, processed and
`kindly supplied by the Tissue Bank of the Plastic Surgery
`Department of the College of Medicine at the University of
`S510 Paulo (USP), Sao Paulo, Brazil. The cells were main-
`tained in DMEM—Fl2 (Dulbecco’s modified Eagle’s medium)
`supplemented with 10% (v/v) heat-inactivated fetal calf serum
`(Cult lab, Brazil), incubated at 37 °C with 5% CO3.
`For adherence assays, cells were seeded (in the absence of
`feeder
`fibroblasts, antibiotics, antimycotics and fetal calf
`serum) into six-well plates at a density of l.O X 106 cells/
`well and grown to confluence in DMEM—Fl2 medium.
`CFAD V. Anacor, |PR2015-01776 ANACOR EX. 2057 - 6/11
`
`
`
`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2057 - 6/11
`
`

`
`L.C. Baeza et al. / Microbes and Infection 9 (2007) 1415-142]
`
`1417
`
`Next, 1.0 X 107 conidia/mL of T. rubrum was added to the ker-
`atinoeytes and incubated for 3 h, 8 h, 24 h and 48 h at 37 °C,
`to follow the processes of adhesion. After the period of incu-
`bation, the medium was discarded, the cells washed with phos-
`phate-buffered saline (PBS), fixed with 4% paraformaldehyde
`and stained by the May—Griinwald (Giemsa) procedure. The
`plates were examined by conventional microscopy to evaluate
`the kinetics of interaction of T. rubrum with keratinocytes.
`
`2.6. RNA extraction fronz keratinocytes
`
`Keratinocytes were plated in 25-cmz tissue culture flasks.
`The conditions of cell culture and infection were as described
`
`above, 24 h of infection being chosen for the RNA extraction.
`After this period, the cells were washed three times in PBS,
`and then incubated in PBS containing trypsin (0.2%) and
`EDTA (0.02%) for total monolayer removal. The cells were
`centrifuged at 5000;; and the pellet was recovered for RNA
`extraction, as described previously. RNA samples for experi-
`ments of dot blot, northern blot and real-time PCR were
`obtained from two independent extractions. Controls were ob-
`tained from the uninfected in vitro cultured keratinocytes.
`
`2.7. Dot blot and northern blot analysis
`
`Serial dilutions of plasmid DNA were vaeuum—spotted on
`nylon membrane and hybridized to the specific cDNAs probes,
`labeled with the Random—Prime DNA Labeling Kit
`(GE
`
`Healthcare). Hybridization was detected by the Gene Images
`CDP—Star Detection Kit (GE Healthcare).
`In the northern
`blot experiments, the RNAs (20 ttg) were fractionated by elec-
`trophoresis in 1.2% agarose—-formaldehyde gels and trans-
`ferred to nylon membrane. RNAs were hybridized to the
`corresponding cDNA probe (Gene Images CDP—Star Detection
`Kit, GE Healthcare). Probes were labeled with the Random-
`Prime Labeling Kit (GE Healthcare).
`
`2.8. Real—time PCR analysis of representative regulated
`genes in T. rubrum
`
`The reaction mixtures contained 2 uL of cDNA, 12.5 ttL of
`SYBR green ROX mixture (Applied Biosystems), and 400 nM
`of each primer, and the volume was brought to 25 ttL with
`nuclease—free water. The reaction program was 50°C for
`2 min, 95 °C for 10 min, and 40 cycles of 95 °C for 15 s and
`the annealing and synthesis at 60°C for 1min. Following
`the PCR, melting-curve analysis was performed, which
`confirmed that the signal corresponded to a single PCR prod-
`uct. Reactions were performed in three PCR repeats with an
`Applied Biosystems 7500 cycler. Data were analyzed by the
`2"AACIi method [13]. The cycle threshold values for the dupli-
`cate PCRs for each RNA sample were averaged, and then
`2”AACT values were calculated (chsl — chitin synthase
`1 was used as the reference). This was followed by normaliza-
`tion to the value for RNA samples from T. rubrum cultured in
`the absence of keratin. A negative-control sample was used
`that contained all
`reagents except T.
`rubrum cDNA and
`
`cDNA obtained from the keratinocyte culture. After 40 rounds
`of amplification, no PCR products were detected in either
`reaction.
`
`3. Results
`
`3.1. Idel'ItlflCatl0)1 of T. rubrum genes with
`a’zfi”erential expressiotz
`
`RDA was performed on the fungus cultured in the absence
`of keratin (driver) and the presence of keratin (tester). Differ-
`ent patterns of DNA amplification were observed after two
`rounds of subtractive hybridization, as shown in Fig. 1.
`A total of 344 clones were successfully sequenced (Table 1).
`The mean size of ESTs was 364 nucleotides. Using the
`BLASTX program, 6.98% of the ESTS corresponded to pro-
`teins of unknown function, with no matches in databases. In
`addition, 94.7% of the ESTS had not been described in T. ru-
`brunz while 5.3% had been.
`
`3.2. Characterization of the subtracted cDNAs from
`T. rubrum cultured in the presence of keratin
`
`The ESTs were classified into seven groups of functionally
`related genes (Table l). The data illustrated the functional di-
`versity of these highly expressed ESTs, denoting particular
`
`506/517
`
`Fig. l. RDA products analyzed by gel electrophoresis. Lanes 1 and 3: products
`of the lirst and second rounds of subtraction, respectively, performed by using
`tester the cDNA obtained from RNA of T. rubrum cultured in the presence of
`keratin. Lanes 2 and 4: products of the first and second rounds of subtraction,
`respectively, performed by using tester‘ the cDNA obtained from RNA of
`T. rubrum cultured in Cove’s medium without keratin. M: molecular markers
`l kb (Invitrogen, CA, USA). Numbers on the right indicate size in bp.
`
`
`
`CFAD V. Anacor, |PR2015-01776 ANACOR EX. 2057 - 7/11
`
`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2057 - 7/11
`
`

`
`I418
`
`L.C. Baeza et ul. / M/('l'0f)L’.Y and ltifection 9 (2007) [4/5-1421
`
`Table l
`
`ESTs with high abundance in T. I‘ltf)I'Iml cultured in the presence of keratin
`E—value
`
`Organism best
`hit/accession number“
`
`Redllilduncy
`
`MIPS category
`
`Gene Product
`
`Transcription
`
`Cell rescue, defense and virulence
`
`Cellular cominunication/signal
`transduction
`Metabolism
`
`Zinc finger protein”
`Transcription factor bZlPh
`Transcription factor homeobox"'°
`Catalase isozyme Pb”
`30 kDa heat shock protein
`G—protein subunit alpha“
`
`Aspergilltis frinzigattis/CAD296()8
`A.speI'gi//tr.r_/imIigarus/XP747348
`Aspergilltls fimzigatus/XP7S2424
`/ljel/omyces capsztlalus/AAN28380
`Trio/zapliytan rubrimz/AAV33735
`Penici/limn chr'y.mgcnum/ABH10690
`
`3E—— I0
`3E—47
`9E—30
`lE—l00
`6E—-l4
`313-95
`
`19
`2
`48
`43
`3
`l 12
`
`Cellular organi7.ation
`Protein synthesis
`Unclassified protein
`
`Probable ATP—dependent RNA helicase DEDl"'°
`5l
`lE—84
`N('llI'()S])()I‘(l cmssa/CAB88635
`Formate dehydrogenase"
`7
`lE—72
`Cocc'itl/aic/es inimitis/EAS37296
`Membrane protein"‘°
`16
`lE—-12
`CI'y]Jf0L'U(.'Cl(.Y IIeqfbt‘mans/AAW4308l
`Nonribosomal peptide synthetase"
`2
`315-33
`Aspergillus fimiigatirs/XP752404
`Conserved hypothetical protein"
`l0
`8E—39
`/l.\'])L’l‘gff/ZIS m'clulans/XP66l692
`Conserved hypothetical protein“
`2
`lE~30
`/lspergil/us /zidtr/ans/XP680743
`Conserved hypothetical protein"
`5
`2E-07
`A.s[w/‘gi//its nitlr//ans‘/XI’66207()
`Hypothetical protein“
`1
`—
`——
`Hypothetical proteinb
`3
`—
`—
`Hypothetical protein!’
`4
`——
`——
`Hypothetical protein”
`3
`._
`—
`12
`—
`—
`Hypothetical protein"
`Hypothetical proteinb
`" Accession number at GenBank (http://www.ncbi.nlm.nih.gov).
`'’ Novel genes detected in T. rulmmz.
`“ Validated up-regulated transcripts.
`
`3.4. Quantitative analysis of genes in T. rubrum by
`t'eal-tiriw PCR
`
`The fungus showed high adhesion to the cell after all the
`periods of time and conidial germination was observed in
`24 h. This time of infection was thus chosen for RNA extrac-
`
`tion during the infection (Fig. 4). To estimate the relative
`transcript
`levels of the differentially expressed products,
`a real—time PCR assay was performed. Fig. 5 shows quantifica-
`tion of the transcript levels of several differentially expressed
`genes. Among the six genes evaluated,
`the catP gene was
`induced 3.8-fold in T. rttbrtmt grown in the presence of keratin
`
`Metabolism
`16.86%
`
`Cellular
`organization
`465%
`
`Protein synthesis
`/-
`0.58%
`
`x Conserved
`hypothetical protein
`4.94%
`
`
`
`Hypothetical
`protein
`6.98%
`
`Transcription
`20.06%
`
`Signal transduction
`32.56%
`
`
`
`Cell defense
`13.37%
`
`Fig. 2. Functional classification of T. rulmmz ESTs derived from RDA exper-
`iment. This classification was based on BLASTX homology of each EST
`against
`the GenBank nr database at a significant homology cttt—off of
`§lE—05 and MIPS functional annotation scheme. Each functional class is
`represented as a color-coded segment and expressed as a percentage of the to-
`tal number of ESTs in each library.
`
`functional categories. The most redundant cDNAs appearing
`during the contact with keratin were as follows: G—protein st1b—
`unit alpha ( gpa), ATP—dependent RNA helicase DEDI (dedl),
`homeobox transcription factor (/ixf), catalase isozyme P (catP),
`zinc finger protein (zfp) and membrane protein (mcmb), as
`shown in Table 1. A reverse cDNA—RDA experiment was
`conducted in which the driver was RNA from keratin culture
`and the tester was RNA from minimal medium culture. A total
`of 33 clones were sequenced, as control. The transcriptional
`profile did not display any similarity with that described for
`T. rztbrtmz cultured in the presence of keratin (data not shown).
`Fig. 2 depicts the classification of 19 clusters of T. I'll/7l‘Lll’Il
`ESTs according to the classification developed at MIPS.
`
`3.3. Confirmatory dtfifercntial expression of T. rubrum
`it/enti_'/ied sequences
`
`To corroborate the RDA findings, we initially performed
`dot blot analysis of T.
`rttbrtmz cDNA—RDA clones. Dot
`blots displayed a differential hybridization pattern when indi-
`vidual clones were hybridized to labeled cDNAs obtained
`from the microorganism cultured in the presence and ab-
`sence of keratin. The level of transcripts corresponding to
`cDNA clones was altered in the presence of keratin, as
`shown in Fig. 3A.
`Northern blot analysis was employed to evaluate the
`expression of some up-regulated genes. The transcripts of
`the genes encoding GPA, CATP, ZFP and MEMB were found
`to have accumulated more in the fungus cultured in the pres-
`ence of keratin (Fig. 3B).
`
`
`
`CFAD V. Anacor, |PR2015-01776 ANACOR EX. 2057 - 8/11
`
`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2057 - 8/11
`
`

`
`L.C. Baeza er al. / Micro/)e.t' um] Infvc1‘i()n 9 (2007) I4/5-I42/
`
`1419
`
`
`
` -
`h.-n
`Fig. 4. Interaction between T. rubrmn and keratinocytes. Cells were seeded
`into six-well plates and incubated with conidia for 2411. The wells were
`washed and stained with the May—Grunwald (Giernsa) for micrographs.
`
`..i
`
`host conditions. Many of the genes found here have already
`been described in orthologous systems and some of them
`have functional roles during the infection process.
`In our study, we sequenced 344 clones, of which 181 were
`ESTs identifying genes encoding proteins involved in tran-
`scription processes and signal transduction. This abundance
`of ESTs for transcription and signal
`transduction proteins
`may be related to fungal growth in the keratin medium. We
`also identified some virulence factors (catalase, 30 kDa heat
`
`[5] constructed a suppres-
`shock protein). Kaufman et al.
`sion—subtractive hybridization (SSH) cDNA library for T.
`menragrop/zyres cultivated on minimal medium with keratin;
`the major up—regulated transcript was thioredoxin, consistent
`with up-regulation of a catalase here.
`On the other hand, Wang et al. [14] constructed 10 different
`T. 1'14/7I‘llI7'l CDNA libraries and obtained 11,085 ESTs. The
`
`identified ESTs encoded putative proteins implicated in pri-
`mary metabolism, gene expression, post-translation processes
`and cell structure. A significant proportion of the identified
`ESTs were matched to genes involved in transcription and sig-
`nal transduction, as found in any eukaryote.
`The G—protein subunit alp/Ia (gpa) transcript was the most
`abundant (112 ESTs), in our experimental conditions, and it
`was up—regulated both during the fungal contact with keratin
`and during the interaction with keratinocytes. The over-
`expression of gpa in T. rubrum in the host-like conditions
`described here strongly suggests that GPA may play an
`
`123455
`A 123456
`“XWIGIII
`”*’@»@0llI
`ded1=L§fifia." ded1.«'.'|'|
`mascot: meuttlt
`catPIllll
`catPll:
`membmeoatu memeeolut
`gpa/)3):
`gpa/tli
`
`wetsuit
`(a) Presence of keratin
`
`W
`
`(b) Absence of keratin
`
`B
`
`catP
`
`
`
`Fig. 3. Validation of the cDNA—RDA results. (A) Dot blot analysis of T.
`rubrmn. DNAs
`of
`individual
`clones were
`prepared
`and
`dilutions
`(1:2000—l:64,000) were blotted (1-6). Panel a: individual clones hybridized
`to the labeled CDNA obtained from T. rubrrmr cultured in the presence of ker-
`atin. Panel b: individual clones hybridized to the labeled eDNA obtained from
`T. rubrmn cultured in the absence of keratin. The clones were as follows:
`G—protein subunit alpha ( gpa), ATP-dependent RNA helicase DEDI ((I(’(/1).
`homeobox transcription factor (Inf), catalase isozyme P (cmP), zinc finger
`protein (zfp), membrane protein (memb), and actin (act) as control. (B) Expres-
`sion patterns of genes obtained by cDNA—RDA analyzed by northern blot of
`total RNA of T. rnbrzmz extracted after culture. in the presence (1) and absence
`(2) of keratin. Total RNA was fractionated on 1.2% formaldehyde~—agarose gel
`and hybridized to the cDNA inserts of gpu, call’, z_/)2, mem/7 and actin (act) as
`the loading control.
`
`and 16.3-fold after infection of keratinocytes, while, the (led!
`gene was induced 1.3- and 8.2-fold, when the fungus was
`grown in the presence of keratin and after infection of kerati-
`nocytes, respectively.
`
`4. Discussion
`
`The initial steps in the pathogenesis of cutaneous infections
`involve the capacity of the infecting microorganism to over-
`come physical and innate resistance factors, allowing initial
`adherence, followed by competition with the normal microbial
`flora and subsequent colonization of the cell surfaces [2]. This
`study is‘ the first to use RDA analysis to characterize changes
`in gene expression after