JP2005503831A - Antimicrobial methods and materials - Google Patents

Abstract

The present invention provides methods for identifying agents that bind to a gene product critical for the survival of a microorganism, preferably a staphylococcal microorganism, including agents that interfere with the function of the gene product.

Description

【００６５】
未知コーディング配列の挿入不活化
不活化は、相同組換えによる標的コーディング配列の５´側半分にプラスミドを組み込むことによって達成された。選択されたコーディング配列の内部断片はPCRによって合成した。増幅された断片の長さは約２５０塩基対ないし約３５０塩基対の間であり、コーディング配列の５´末端を含むものであった。増幅で用いたプライマーは、PstI制限部位増幅された断片の１つの端部に付加し、SacI制限部位を増幅された断片の他の端部に付加するようにさらなるヌクレオチドを含むものであった。プライマーは表１に示す。付加された制限部位により、増幅された断片の、温度感受性シャトルベクターpSPT２４６への連結が可能となった。pSPT264は、pRN8103およびpSP64-PolyAを連結することによって構築した。pRN8103温度感受性複製ベクターは、ユニークなEcoRI制限部位を含み、該ベクターはイー・コリでは複製することができない。pRN8103はNovickら, (J. Mol.Biol., 192, 209-220 (1986))に記載されている。Promega Corp.（Mabison, WI）から入手したpSP64-PolyAベクターはイー・コリでは複製するが、エス・アウレウスでは複製しない。pSP64-PolyAもまたユニークなEcoRI制限部位を含む。イー・コリ／エス・アウレウスシャトルベクターは、各ベクターをEcoRIで消化し、２つのベクターを一緒に連結し、次いで、該DNAをイー・コリに形質転換することによって構築した。得られたシャトルベクターをpSPT264と命名した。
【００６６】
組換えプラスミド（すなわち、増幅された断片を含むpSPT246）を用いてイー・コリを形質転換し、それを単離し、次いで、エレクトロポレーションによって（Kreiswirthら, Nature, 305, 709-712 (1983)によって記載された）エス・アウレウスRN4220に移した。形質転換体は、テトラサイクリン（１０μｇ／ｍｌ）を含有する栄養寒天プレート上で許容される温度（３０℃）でインキュベートすることによって選択した。正しいプラスミドの存在はPCRによって確認した。
【００６７】
正しいプラスミドを持つ１つのクローンを、テトラサイクリン（１０μｇ／ｍｌ）を含む栄養寒天上で３２℃にて一晩増殖させて、該プラスミドおよび選択された染色体対立遺伝子の間の組換えを行った。組換え体につき選択するために、次いで、該細菌を、テトラサイクリンを含まないブレイン・ハート・インフュージョン（BHI)ブロス中で許容されない温度（４３℃）にて１８時間増殖させ、続いて、５μｇ／ｍｌテトラサイクリンを含有するBHIブロスに１：１０希釈した。細胞を４３℃にて一晩インキュベートした。次いで、細菌培養を希釈し、５μｇ／ｍｌテトラサイクリンを含有する栄養寒天プレート上に広げ、４３℃にて一晩インキュベートした。該プラスミドは４３℃においては複製できないので、染色体に組み込まれたプラスミドを持つ細胞のみがテトラサイクリン耐性であり、コロニーを形成する。許容されない温度で出現するミクロコロニーも考えられる。というのは、それらは、重要である（例えば、臨界的）が、増殖には必須ではないコーディング配列において突然変異を示し得るからである。
【００６８】
該プラスミドは染色体中の他の部位においては組み込まれる頻度が低く、かくして、標的コーディング配列が必須である場合のみテトラサイクリン耐性クローンが出現した。従って、４３℃における各選択からの１０コロニーを、PCRによって、選択された標的コーディング配列へのプラスミドの特異的組み込みにつきテストした。ベクターDNAに結合する１つのプライマー、および染色体中の標的コーディング配列の上流に結合する第二のプライマーよりなるプライマー対をPCR増幅に用いた。該プライマー対は、介入する染色体−ベクター領域を増幅させ、もし該ベクターが予測される位置に組み込まれた場合のみ、増幅されたDNA断片が生産される。バンドの不存在は、ベクターが組み込むことができず、該コーディング配列が必須であることを示唆する。典型的には、テストしたコロニーの全てが特異的組換え体であるか、あるいはいずれの該組換え体ではなかった。組換え体が見出されない場合、該標的コーディング配列は必須と考えられる。多数の標的コーディング配列（必須および非必須双方）については、全選択手法を繰り返した場合に、同一結果が得られた。
【００６９】
このプロトコルは、同定された４９２の未知の完全なまたは部分的なコーディング配列のうち約３００を分析するのに首尾よく用いられた。分析したコーディング配列のうち、２６は臨界的なように見え、さらに、後記するごとく分析した。
【００７０】
実施例２
必須エス・アウレウスコーディング配列のクローニングおよびイー・コリにおける発現
発現系およびクローニング手法の概観
エス・アウレウス蛋白質の過剰発現は、QiagenタイプATG発現系（Qiagen Gmbh, Santa Clara, CA）を用いて達成される。この系は、その遺伝子型がNal^s、Str^s、rif^s、lac^-、ara^-、gal^-、mtl^-、F^-、recA⁺、uvr⁺としてQiagenによって記載されているイー・コリ株「M15」を利用する。２つの複製適合ベクターであるpREP4およびpQE-60（各々、Qiagenから入手）は、当該手法の間にM15株に導入される。あるいは、pQE-70をpQE-60の代わりに用いることができる。pREP4ベクターは、ラクトース（LacI）リプレッサー蛋白質につきコードするlacI遺伝子を含むpACYC-由来ベクターであり、該ベクターはカナマイシン薬物耐性をコードする。発現ベクターpQE-60は、修飾されたT5ファージプロモーター、強力なリボソーム結合部位（RBS）、および発現されるべき特異的エス・アウレウスのコーディング配列のコーディング配列を含むpBR322-由来ベクターである。T5プロモーターの修飾は、LacIリプレッサーによる該プロモーターの結合および調節のためのオペレーターの置き換えを含む。発現の誘導は、対数期培養へのIPTG（イソプロピルチオ−β−Ｄ−ガラクトシド）の添加によって行われる。
【００７１】
一般的なクローニング戦略は、まず、コーディング配列の５´および３´末端に対するPCRプライマーを用い、エス・アウレウスのゲノムDNAからの特異的コーディング配列を増幅させることである。該PCRプライマーは、各々、コーディング配列の５´および３´末端においてNcoIおよびBglII制限部位を付加するように設計されている。コーディング配列は、いずれのNcoIまたはBglII制限部位も含むべきではない。もしそのような部位が存在すると、それは、当業者に知られた部位特異的PCR突然変異誘発手法を用いて排除する。別法として、異なる制限部位、例えば、BamHI制限部位をBglII制限部位の代わりに用いる。増幅されたエス・アウレウスのコーディング配列をpCR-2.1（Invitrogen, Carlsbad, CA）に連結し、当該分野で知られた技術を用いてイー・コリに形質転換される。PCR増幅またはベクター制限分析によって、コロニーをコーディング配列の存在についてスクリーニングする。クローンはランダムに選択し、挿入DNAのヌクレオチド配列、すなわち、エス・アウレウスのコーディング配列を、挿入の真正を確認するように測定する。
【００７２】
所望のコーディング配列含むpCR-2.1ベクターをNcoI/BglIIで消化し、コーディング配列を単離し、pQE-60の対応するNcoI/BglII制限部位に連結する。連結混合物を用いて、ベクターDNAを、pREP4ベクターを含むM15株にエレクトロポレーションにより入れる。得られた形質転換体はPCRまたは制限分析によってスクリーニングする。候補を震盪フラスコ中で増殖させ、SDS-PAGEまたはウエスタン分析によって分析して、適切なサイズを有する蛋白質バンドの過剰発現につきスクリーニングする。抗His抗体（Invitrogen）をウエスタン分析で用いる。単一候補を、各コーディング配列によってコードされた蛋白質の過剰発現および単離につき選択する。
【００７３】
培養および培地
イー・コリにおける組換えプラスミドを含む細胞のクローニングおよび維持のための培地は、適当な抗生物質（１００μｇ／ｍｌアンピシリン、２５μｇ／ｍｌカナマイシン）を補足したLBである。エス・アウレウスをミューラー・ヒントン培地中で増殖させた。製造業者の指示に従い、コンピテントINVF'α細胞（Invitrogen, Carlsbad, CA）を用いる。M15 pREP-4株はQiagenから購入した。細胞のエレクトロポレーションでは、SOC培地を用いた。LBおよびSOC培地はSambrookら（Molecular Cloning: A Laboratory Manual., Cold Spring Harbor Laboratory Press, pp. Al-A4 (1989))に記載されている。ミューラー・ヒントン培地はAtlasら, Handbook of Microbiological Media, CRC Pressに記載されている。
【００７４】
pQE60発現ベクターの設計
T5プロモーター、RBS、ATG開始コドン（太文字）、NcoI制限部位（下線）、BglII制限部位（下線）、6Hisタグ（二重下線）、およびTAA停止コドン（太文字）を含むpQE-60DNA配列の一部を示す（配列番号：89）：
【００７５】
【００７６】
【化１】

【００７７】
エス・アウレウスのコーディング配列は、適合するイン−フレームNcoIおよびBglII制限部位を含むようにPCRで修飾する。
【００７８】
プライマーの設計
エス・アウレウスコーディング配列の５´部分に対するプライマーの設計用の一般式は通常５´-CCATGGGAN_20-30であり、３´プライマーについての一般式は通常５´-AGATCTN_20-30である。これらのプライマーはNcoIおよびBglII制限配列を付加する。５´配列の最初の「N」ヌクレオチドは、そのATG開始後のエス・アウレウスコーディング配列の第二のアミノ酸のコドンに対応する。３´プライマーの最初の「N」ヌクレオチドは、エス・アウレウスコーディング配列の停止コドンに先行するコドン中の第三のヌクレオチドに対応する。プライマーに含まれるヌクレオチドの数は特異的DNA配列に応じて変化するが、典型的には２０ないし３０塩基の範囲である。該プライマーはリン酸化される。いくつかのコーディング配列を増幅するのに用いることができるプライマーの例は表２に示す。
【００７９】
【表２】

【００８０】
エス・アウレウスのゲノムDNAの調製
(S.Arvidson, Karolinska Instituteから入手した)株ＩＳＰ３を用いて、１０ｍｌのミュラー・ヒントンブロスを接種する。３７℃における一晩の増殖の後、１．５ｍｌの培養をエッペンドルフ試験管中にペレット化し、次いで、４００μｌのＴＥ（pH8.0）に再懸濁する（Sambrookら（Molecular Cloning: A Laboratory Manual., Cold Spring Harbor Laboratory Press, p. B.20 (1989)）。５０μｌのリソスタフィン溶液（１０ｍｇ／ｍｌ）の添加に続き、細胞を３７℃にて１時間インキュベートする。７０μｌの１０％ＳＤＳおよび１０μｌのプロテイナーゼＫ（２０ｍｇ／ｍｌ）を添加し、３７℃にてさらに１時間インキュベーションを継続する。１００μｌの５ｍＬ NaClの添加の後、細胞懸濁液を撹拌し、１０％臭化ヘキサデシルトリメチルアンモニウム、０．７Ｍ NaClを含有する溶液（CTAB/NaCl）８０μｌを添加する。細胞を撹拌し、次いで、６５℃にて１０分間インキュベートする。等容量の２５：２４：１フェノール：クロロホルム：イソアミルアルコールの添加に続き、細胞を撹拌し、５分間遠心する。次いで、水性層を新鮮な試験管に移し、白色CTAB/NaCl界面を得る。抽出は反復し、水性層を再度新鮮な試験管に移す。等容量のイソプロパノールの添加に続き、該試験管を軽く混合し、糸状沈殿が形成されるようにする。小さなフック形状のパスツールピペットを用いて、沈殿を温和に除去し、それを、１ｍｌの７０％エタノールを含有するもう１つの試験管に移す。該試験管を遠心し、得られたペレットを７０％エタノールで１回洗浄する。乾燥の後、DNAペレットを１００μｌの水に再懸濁させ、当該分野で公知の技術を用いて回収されたDNAの濃度を測定する。
【００８１】
PCR増幅
Perkin-Elmer Cetus GeneAmp 9600または2400熱サイクラー（Perkin-Elmer, Norwalk, CT）いずれかを用いて、PCR反応を行う。デオキシヌクレオチドミックスおよびPfu DNAポリメラーゼはStratagene（La Jolla, CA）から購入する。AmpliTaq GoldキットはPerkin Elmerから購入する。長い鋳型増幅のためのPCR合成プロトコルは、以下の通りである：１μｇのエス・アウレウスゲノムDNA、１０μｌの１０×反応緩衝液（１５ｍｍ MgCl₂）、５００ｍｇの各プライマー、１６μｌの１．２５ｍＭ dNTP、１μｌのAmpliTaq Gold、および１００μｌまでの水をPCRミクロ試験管当たり加える。５分間の９５℃、続いて、３０秒間の９４℃、１分間の５０℃および３分間の７２℃の３５サイクル、５分間の７２℃における延長、および最後の４０℃保持のサイクルプログラムを用いる３５サイクルにて、DNAを増幅させる。合成反応の１０μｌのアリコットを１．２％アガロースゲルに負荷し、合成された断片の存在およびサイズを確認する。PCR産物は、多重PCR反応を組合わせ、DNAをEtOH沈殿させ、次いで、１．２％アガロースゲルから所望の断片を切断することによって生じる。Amicon Ultrafree-DA抽出フィルター（Millipore Corp., Bedford, MA）を用い、DNAをアガロースから単離する。フィルターは製造業者の指示に従って用いる。
【００８２】
連結および形質転換
pQE-60ベクターおよびエス・アウレウスコーディング配列を含むpCR2.1ベクターをNcoIおよびBglII制限酵素で消化する。pQE-60ベクター断片およびエス・アウレウスコーディング配列をアガロースゲルから単離する。２つのDNAを連結させ、pREP-4を含むエレクトロコンピテントM15細胞に形質転換し、アンピシリンおよびカナマイシンを補足した。LD寒天上で平板培養する。リガーゼはBioLab（Beverley, MA）から購入し、製造業者の指示に従って用いる。連結されたDNAのM15 pREP-4細胞へのエレクトロポレーションは、Bio-Rad Geneパルサー（Hercules, CA）を用いて行う。A₅₅₀において１の光学密度を持つ１リットルの細胞からコンピテント細胞が調製される。細胞を冷却し、順次、１リットルおよび０．５リットル氷冷滅菌水で洗浄する。細胞を２０ｍｌの氷冷滅菌グリセロールに再懸濁させ、再度遠心し、２ないし３ｍｌの冷滅菌１０％グリセロールの最終懸濁液に入れる。５０μｌの細胞を５μｌ以下の連結DNAと混合する。細胞／DNA混合物をエレクトロポレーションキュベットに移し、２５μＦ，２．５ｋＶの設定、および２００Ωに設定されたパルスコントローラーでパルスを与える。次いで、１ｍｌのSOC培地を加える。細胞を３０℃にてインキュベートし、選択培地上に置く。
【００８３】
形質転換からのいくつの得られたコロニーをランダムに選択し、Qiagenから購入したMiniprepまたはMaxiprepキットを用いてベクターDNAを単離する。ベクターDNAは製造業者の指示に従って単離する。候補は制限酵素消化によってスクリーニングする。制限酵素はNew England BioLab（Beverly, MA）から購入する。制限酵素は製造業者の指示に従って用いる。
【００８４】
発現条件
発現培養は、アンピシリンおよびカナマイシンを含有するLBプレート上で画線培養する。単一コロニー単離体を用いて、アンピシリンおよびカナマイシンを補足した５０ｍｌのLB培地に接種し、所望の温度にて一晩増殖させる。0.50A₅₅₀/mlにおける適当な容量の同一培地への継代培養に続き、３．０のA₅₅₀に達するまで、同一温度にて激しくエアレーションしつつ培養を増殖させる。IPTGの添加によって、培養を１ｍＭの最終濃度まで誘導する。培養アリコットを、SDS-PAGEまたはウエスタン分析のために、誘導後０、２および４時間に取り出す。細胞を４および６時間の間に蛋白質単離のために収穫する。金属キレートアフィニティークロマトグラフィー精製システム(QIA Express、Qiagen)を用い、蛋白質を単離する。
【００８５】
実施例３
抗微生物剤についてのスクリーニングにおける必須コーディング配列産物の使用
個々の精製された蛋白質(すなわち、標的蛋白質)を試料と合わせ、標的蛋白質に結合するリガンドにつきスクリーニングする。スクリーニングするのに用いる方法はHughesらの米国特許第5,783,397号に記載されている。スクリーニングは、Cetek Corporation、Narlborough、MAによって行われる。
【００８６】
実施例４
イー・コリにおける発現用のエス・アウレウスウリジル酸キナーゼコーディング配列のクローニング
プラスミドpREP4を含むイー・コリ株M15における組換えウリジル酸キナーゼの生産用の発現ベクターpQE60に、ウリジル酸キナーゼにつきコードするエス・アウレウスpyrHコーディング配列をクローンする。pyrHコーディング配列のクローニングは、鋳型としてエス・アウレウスのゲノムDNAを用いる、２つのオリゴヌクレオチドプライマー５´CCCGGGCCATGGCTCAAATT（配列番号：90）および５´GGGCCCAAGCTTAGTGATGG（配列番号：145）でのPCRによるものであった。PCR産物を制限酵素NcoIおよびHindIIIで処理し、アガロースゲル電気泳動によって精製し、NcoIおよびHindIIIで消化したpQE60に連結する。連結混合物を、pREP4を含むM15細胞に形質転換し；形質転換体を選択し、制限酵素分析およびDNA配列決定によって、pyrHコーディング配列のヌクレオチド配列を確認した。イー・コリにおけるエス・アウレウスウルジル酸キナーゼの生産用の得られたプラスミドをpQE60-UMKと命名した。DNAおよびプラスミド調製、制限酵素処理、連結、および形質転換についての手法はSambrookら（Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)）に記載されているものに従った。
【００８７】
組換えウリジル酸キナーゼのヌクレオチドおよびアミノ酸配列を図３に示す。Ni-キレーティングクロマトグラフィーでの精製のために、６つのヒスチジン残基をC-末端に付加する。クローニング用のNcoI部位およびベクター配列の使用のため、開始メチオニン後にアラニン残基を付加し、ヒスチジン残基に先立ってアルギニンおよびセリン残基を付加する。
【００８８】
実施例５
組換えエス・アウレウスウルジル酸キナーゼの産生
N15（pQE60-UMK）と命名された生産株を、３０℃にてNS86培地で約１のA₅₅₀まで増殖させた。NS86倍地は１リットルの水中の２．６ｇのK₂HPO₄、１０．９ｇのNaNH₄HPO₄-4H₂O、２．１ｇのクエン酸、0.67gの（NH₄)₂SO₄、０．２５ｇのMgSO₄-7H₂O、１０．４ｇの酵母エキスおよび５ｇのグリセロールである。NS86培地は１００μｇ／ｍｌのアンピシリンおよび２５μｇ／ｍｌのカナマイシンを補足したものであった。凍結保護剤として２０％グリセロールを添加した凍結アンプルを調製し、液体窒素中で保存した。
【００８９】
アンプルからの０．１ｍｌ解凍細胞の５０ｍｌのNS86倍地への接種によって種培養を調製し、３０℃で一晩増殖させ、これを用いて１００ｍｌのMIM培地を接種した。MIM培地は、A₅₅₀0.1までの１００μｇ／ｍｌアンピシリンおよび２５μｇ／ｍｌカナマイシンを含有する１リットルの水中の３２ｇのトリプトン、２０ｇの酵母エキス、６ｇのNa₂HPO₄、３ｇのKH₂PO₄、０．５ｇのNaClおよび１ｇのNH₄Clである。細胞を３０℃にて一晩A₅₅₀7-8まで増殖させ、１０リットルの発酵で用いた。
【００９０】
種培養を用いて、A₅₅₀0.1までMIM培地を含む１０リットルのファーメンター（New Brunswick Microgens）を接種した。細胞が３０℃にて約１のA₅₅₀の密度まで増殖すると、イソプロピル−β−Ｄ−チオガラクトシドを１ｍＭまで添加して、組換え蛋白質の発現を誘導した。細胞を誘導２時間後に収穫し、凍結保存した。１０リットルの発酵から生じた平均量のウリジル酸キナーゼは１７０ｍｇ／ｌにおいて見積もり、これは合計細胞蛋白質の約２０ないし２５％に対応する。
【００９１】
実施例６
組換えエス・アウレウスウリジル酸キナーゼの精製
１０リットルの発酵からの凍結細胞を解凍し、２００ｍｌの冷溶解緩衝液（５０ｍＭトリス、２２℃においてpH7.8、500mM NaCl、１０％グリセロール、２５ｍＭイミダゾール、５ｍＭ ２−メルカプトエタノール、0.1mg/ml DNase）と混合した。ペレットをホモゲナイズして均一な懸濁液が得られ、これをRainieホモゲナイザーで２回処理して、細胞を溶解させた。溶解した細胞を３５，０００×ｇにて７５分間遠心し、上澄み液体を順次Nalgene 0.45ミクロンおよび0.2ミクロンフィルターを通して濾過して、カラムクロマトグラフィーに先立って粒状物を除去した。
【００９２】
カラムクロマトグラフィーは、Qiagen Ni-NTA Superflow樹脂を充填した2.6cm×6.7cmカラムを用いて４℃で行った。カラムを３ベッド容量の水で洗浄し、２５ｍＭイミダゾールを含有する３ベッド容量の平衡緩衝液（５０ｍＭトリス、２２℃にてpH7.8、500mM NaCl、１０％グリセロール、５ｍＭ ２−メルカプトエタノール）で平衡化した。濾過した上澄みを１時間当たり３ベッド容量の速度でカラムに適応した。負荷の後、２８０ナノメートル（ｎｍ）における吸光度がベースラインの５０％まで減少するまで、２５ｍＭイミダゾールを含む平衡緩衝液でカラムを洗浄し、続いて、６ないし７ベッド容量の平衡緩衝液＋４０ｍＭイミダゾール、次いで、６ないし７ベッド容量の平衡緩衝液＋５０ｍＭイミダゾールで洗浄した。平衡緩衝液＋３００ｍＭイミダゾールにて１時間当たり２ないし３ベッド容量の速度で結合したウリジル酸キナーゼを溶出させ、４つの別々の画分に回収し、これをプールし、３倍希釈して蛋白質濃度を低下させ、５０ｍＭトリス、２２℃にてpH7.8、500mM NaCl、１０％グリセロール、５ｍＭ ２−メルカプトエタノールに対して透析した。透析したプールをさらに使用するまで凍結保存した。
【００９３】
この単離により９５ないし９８％の純度で約７００ｍｇの組換えウリジル酸キナーゼ蛋白質が得られた。Ｎ−末端の配列決定により、Ｎ−末端メチオニンが存在することが示され、これは宿主メチオニンアミノペプチダーゼの活性によるものと予測される。
【００９４】
実施例７
ウリジル酸キナーゼ活性についての酵素アッセイ
ウリジル酸キナーゼは、ホスホリル基のATPからUMPへの移動を触媒して、UDPを形成する。細胞中においては、UDPは、RNAおよび合成を含めたいくつかの代謝経路において基質／前駆体である。分光測定アッセイは、ピルビン酸キナーゼおよび乳酸デヒドロゲナーゼを用いてウリジル酸キナーゼ反応物をNADH酸化にカップリングさせることによって確立され、これは、結局は、ウリジル酸キナーゼ反応の産物をラクテートおよびNAD^＋に変換する。NADH酸化は、３４０ｎｍにおける吸光度の減少によってモニターした。５ｍＭの最終濃度におけるEDTAを用いてアッセイを停止した。このアッセイは、ウリジル酸キナーゼを阻害する剤につきスクリーニングするために９６−ウエルマイクロタイタープレートにおいて高スループット様式で最適化した。ピルビン酸キナーゼおよび乳酸デヒドロゲナーゼカップリング酵素についての第二のアッセイも開発して、一次カップリングしたアッセイで検出された剤の特異性をテストした。
【００９５】
カップリングされたアッセイを以下に示す。
【００９６】
【数１】

【００９７】
略語：
ATP/ADP−アデノシン５´三リン酸／アデノシン５´−二リン酸
UMP/UDP−ウリジン５´一リン酸／ウリジン５´二リン酸
PEP−ホスホ（エノール）ピルベート
NADH/NAD+−還元された／酸化されたニコチンアミドアデニンジヌクレオチド
ウリジル酸キナーゼカップリングアッセイは２００μｌの最終容量中に以下の試薬を含む：アッセイ緩衝液（50mM HEPES、pH7.5、100mM KCl、2mM MgCl_２）、1mM UMP、2mM ATP、0.22mM NADH、2mM PEP、3.2ユニットのピルビル酸キナーゼ（Sigma, St.Louis, MO）、４ユニットの乳酸デヒドロゲナーゼ（Sigma）および１０ｎｇのウリジル酸キナーゼ。該アッセイは２５℃で行い、１または３時間の動的モードにて１５秒間の間隔でモニターして３４０ｎｍで吸光度が減少した。
【００９８】
本明細書中で引用した、例えば、GenBank受託番号およびEMBL受託番号を含めた、全ての特許、特許出願、刊行物、および核酸および蛋白質データベースエントリーの完全な開示は、あたかもここに取り込むごとくここに引用して一体化させる。本発明の種々の修飾および変形は、本発明の範囲および精神を逸脱することなく当業者に明らかであり、本発明は本明細書中に記載した例示的具体例に限定されないと理解されるべきである。
【００９９】
【化２】

【図面の簡単な説明】
【０１００】
【図１ａ−ｚ】２６のエス・アウレウスコーディング配列のヌクレオチド配列（配列番号：1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,109,113,117,121,125,129,133,137および141）、各コーディング配列によってコードされたペプチドの予測される配列（各々、配列番号：2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,110,114,118,122,126,130,134,138または142）、および温度感受性プラスミドへの挿入のための断片を調製するために用いるプライマー対（配列番号：35-68,111-112,115-116,119-120,123-124,127-128,131-132,135-136,139-140および143-144）。各コーディング配列における２つの下線を施した配列は、コーディング配列の下方にリストしたプライマーに対応する。
【図２ａ−ｉ】イー・コリにおける発現用にクローンすべき９つのエス・アウレウスコーディング配列の各々のヌクレオチド配列（配列番号：69,71,73,75,77,79,81,83および85）、適当な発現プラスミドへの挿入後に各コーディング配列によってコードされたペプチドの予測される配列（各々、配列番号：70,72,74,76,78,80,82,84および86）、および増幅によってエス・アウレウスコーディング配列をクローンするのに用いるプライマー対の配列（配列番号：91ないし108）。各プライマー対の頂部プライマーおよび底部プライマーは、各々、順方向プライマーおよび逆方向プライマーである。配列番号：69、73、75、77および79における下線を施したATGGは、発現ベクターpQE-60へのクローニング用の順方向プライマーによってコーディング配列付加されたNcoI制限部位の一部の位置を示す。配列番号：69,71,73,75,79,81,83および85における下線を施したAGATCTは、逆方向プライマーによってコーディング配列に付加されたBglII制限部位の位置を示す。配列番号：77における下線を施したGGATCTは、ベクターの消化されたBglII制限部位と、増幅された断片の消化されたBamHI制限部位の連結の位置を示す。
【図３】エス・アウレウスウリジル酸キナーゼのヌクレオチド配列（ハ列番号：８７）および予測されるアミノ酸配列（配列番号：88）。[0001]
Staphylococcus Aureus, Staphylococcus Aureus, the most important human pathogen, is a tough gram-positive bacterium that colonizes most human skin. The strain of staphylococci that produces coagulase is named S. aureus; the other clinically important coagulase-negative staphylococci are S. epidermidis and S. saprophyticus. is there. When the skin or mucosal membrane barrier is disrupted, staphylococci can cause localized superficial infections that are usually harmful and self-limiting. However, when staphylococci enter the lymphatic system and blood, bacteremia, including endocarditis, arthritis, osteomyelitis, pneumonia and abscesses, septic shock, and severe metastases in virtually all organs Potentially serious complications such as sexually transmitted infections can result. Certain strains of S. aureus produce toxins that cause skin erythema, food poisoning or multisystem dysfunction (as in toxin shock syndrome). Together, S. Aureus and S. epidermidis are the most common cause of non-urinary nosocomial infections in US hospitals. They are the most frequently isolated pathogens in both primary and secondary bacteremia and in skin and surgical wound infections. See generally Harrison's Principles of Internal Medicine, 13th edition, edited by Isselbacher et al., McGraw-Hill, New York (1994), especially pages 611-617.
[0002]
Transient colonization of the nose by S. aureus is observed in 70 to 90 percent of people, of which 20 to 30 percent retain bacteria for a relatively long period. Independent colonization of the perineal area occurs in 5 to 20 percent of people. The higher carrier rate of S. aureus has been documented in people with atopic dermatitis, hospital employers, inpatients, patients whose care requires frequent skin punctures, and intravenous drug abusers .
[0003]
Infection with staphylococci results from a combination of bacterial virulence factors and reduced host defense. Important microbial factors include the ability of staphylococci to survive under harsh conditions, its cell wall components, the production of enzymes and toxins that promote tissue invasion, its ability to persist intracellularly in certain phagocytes, and Including its ability to acquire resistance to microbial agents. Severe host factors include the removal of intact mucosal skin barrier, the appropriate number of functional neutrophils, and foreign or dead tissue.
[0004]
S. aureus cell wall components include large peptidoglycan complexes that impart rigidity to the organism, and can bind to peptidoglycans across unfavorable osmotic conditions, together with the outermost part of the cell, and be released in soluble form. It is possible to survive under the conditions of unique tycoic acid linked to peptidoglycan and protein A. Proteins named femA and femB are involved in the formation of peptidoglycan pentaglycine cross-bridges in the cell wall and are factors in methicillin resistance (Berger-Bachi et al., Mol. Gen. Genet., 219, 263-269 ( 1989)). S. aureus also has specific receptors for laminin and fibronectin that can mediate the spread of organisms through the bloodstream to other tissues. Both peptidoglycan and tecoic acid can activate the complement cascade via alternative pathways. S. aureus also appears to activate tissue factor in the coagulation pathway.
[0005]
Certain enzymes produced by S. Aureus can play a role in virulence. Catalase can break down hydrogen peroxide and protect the organism during phagocytosis. Coagulase exists in both soluble and cell-bound forms and causes plasma to clot due to the formation of thrombin-like substances. The high correlation between coagulase production and virulence suggests that this substance is important in the pathogenesis of staphylococcal infections, but its exact role as a determinant of pathogenesis has not been determined. Many strains also produce hyaluronylase, an enzyme that can degrade hyaluronic acid in the connective tissue matrix and promote the spread of infection. Trepsin-like proteases from several strains can enhance influenza virus infection by proteolytic cleavage into active fragments of the viral precursor hemagglutination layer and contribute to the prevalence of such co-infections. S. aureus produces a number of extracellular exotoxins that have been associated with disease processes. Exfoliatin toxins A and B, staphylococcal enterotoxin and toxin shock syndrome detoxin, TSST-1, activates T cells and monocyte macrophages, leading to the amount of toxin formed, host immune status, and toxin circulation Depending on their access, they belong to an increasing family of microbial superantigens that result in the production of cytokines that mediate local or systemic effects. Exforitatin toxin mediates dermatological manifestations of staphylococcal burn-like skin syndrome and vacuolar impetigo. These toxins cause intraepidermal cuts of the skin in the granule layer, leading to blister formation and nakedness. Seven distinct enterotoxins (A, B, C1, C2, C3, D and E) have been associated with food poisoning by S. aureus. These toxins seem to enhance intestinal peristalsis and induce vomiting by direct effects on the central nervous system. Toxin shock syndrome (TSS) is most commonly mediated by PSST-1 present in 5-25% of S. aureus clinical isolates. TSS is also less frequently mediated by enterotoxin B and, rarely, enterotoxin C1.
[0006]
S. aureus produces other toxins whose role in virulence is poorly understood. Four different red blood cell hemolysin have been identified, named alpha, beta, gamma and delta and Takanobu. When injected subcutaneously in animals, alpha toxins cause skin necrosis, while delta toxins inhibit water absorption in the intestine and play a role in acute watery diarrhea observed in some cases of staphylococcal infection. Can play. Leukocidin dissolves the granulocyte and macrophage membranes by creating membrane pores that are permeable to cations.
[0007]
The agr, xpr, sae and sar coding sequences have been identified as being involved in the regulation of staphylococcal exotoxins. See US Pat. No. 5,587,285 and International Patent Publications WO96 / 10579 and WO97 / 11690. Interesting is the report in WO 97/11690 for screening for inhibitors of these regulatory systems.
[0008]
Staphylococci can enter the skin or mucous membranes through hair follicles and sebaceous glands or areas that have been traumatic and blocked by burns, wounds, wear, insect bites or dermatitis. Staphylococci are often colonized with prosthetic devices and intravenous catheters; S. aureus infection of vascular access sites is a major cause of morbidity and mortality among patients undergoing hemodialysis. Lung colonization and invasion can occur with endotracheal intubation or when lung clearance mechanisms are reduced, for example, after viral infection, after aspiration, or in patients with cystic fibrosis. Mucosal damage to the gastrointestinal tract following cytotoxic chemotherapy or radiation therapy is a predisposition to entry from that site.
[0009]
Once the skin or mucous membrane is destroyed, local bacterial growth is accompanied by inflammation, neutrophil accumulation, tissue necrosis, thrombus formation and fibrin deposition at the site of infection. Later, fibroblasts create a relatively non-vascular wall around that area. If the host mechanism does not involve skin or submucosal infection, it can enter the staphylococcal lymphatic system and the bloodstream. Common sites of metastatic spread include the lung, kidney, heart valve, heart muscle, liver, spleen, bone and brain.
[0010]
Bacteremia caused by S. aureus can be either extravascular (cutaneous injection, bums, vesicles, osteomyelitis, arthritis) or intravascular lesions (intravenous catheter) dialysis access site, intravenous drug abuse, It can be from any local infection. The disease usually progresses slowly with wasting fever and metastatic abscess formation. Rarely, patients with bacteremia die within 12 to 24 hours with high fever, tachycardia, cyanosis and vascular collapse. Disseminated intravascular coagulation can result in a disease resembling Neisseria meningitidis.
[0011]
The main complication of S. aureus bacteremia is endocarditis. S. aureus is the second most common cause of endocarditis and the most common cause of drug addiction. The disease is typically acute and is accompanied by high fever, progressive anemia and frequent embolic and extracardiac septic complications. Annulus and myocardial abscess are common. The mortality rate is 20-30%.
[0012]
Staphylococcal scalded skin syndrome (SSSS) is systemic exfoliative dermatitis, a complication of infection by S. aureus exfoliateton toxin producing strain. The disease typically occurs in newborns (Litter disease) and children under 5 years of age. Scarlet fever-like rashes begin in the area around the mouth, become generalized across the trunk, and finally show desquamation. The disease consists of eczema alone (staphylococcal scarlet fever), the development of large flaccid blisters that can be localized or generalized. The blisters rupture, resulting in naked skin that resembles a red burn. Most adults with SSSS are immunosuppressed or have renal failure. Blood cultures are frequently positive and have a high mortality rate.
[0013]
This toxin shock (TSS) is a multisystem disease mediated by toxins produced by certain strains of S. aureus (generally TSST-1, and to a lesser extent enterotoxins B and C1). is there. It was first mentioned in children, but in 1980 it became prevalent among menstruating young women. Diagnosis of TSS is evidence of high fever, a widespread rash with desquamation on the palms and soles over the course of one or two weeks, hypotension that can be orthostatic, and evidence of involvement in more than two organ systems. Based on clinical criteria including. Such involvement is commonly associated with gastrointestinal dysfunction (vomiting and diarrhea), kidney or liver dysfunction, mucosal hyperemia, thrombocytopenia, muscle pain with elevated creatine phosphokinase (CK) levels, and normal cerebrospinal spinal cord Includes disorientation with fluid testing. The death rate of TSS is 3%.
[0014]
S. aureus causes nearly 3 percent of community-acquired bacterial pneumonia. This disease occurs sporadically, except during an influenza epidemic, when staphylococcal pneumonia is relatively more common, but still less frequently than pneumococcal pneumonia. Primary staphylococcal pneumonia in infants and children is frequently present with high fever and cough. Multiple thin wall abscesses are seen on chest x-rays and have common empyema formation. In older children or healthy adults, staphylococcal pneumonia is commonly preceded by influenza-like respiratory infections. The start of staphylococcal involvement is sudden high fever with chills, progressive dyspnea, cyanosis, cough, pleuritic pain and sometimes blood clots. Staphylococcal pneumonia is more frequent in patients with cystic fibrosis, intubated patients in the intensive care unit, and debilitated patients who tend to suck.
[0015]
S. aureus is responsible for most cases of acute osteomyelitis. The disease is most common in people under 20 years of age, but it is increasingly prevalent, especially in adults over the age of 50 with spinal involvement. The main entrance is not frequently confirmed, but many patients give a history of previous trauma to the relevant area. Once established, the infection spreads through the bone to the periosteum or along the bone marrow cavity. Rarely, it penetrates the joint capsule and causes purulent arthritis. Osteomyelitis in children exists as an acute process and can begin suddenly with chills, high fever, nausea, vomiting, and progressive pain at the site of bone involvement.
[0016]
S. aureus causes 1 to 9 percent cases of bacterial meningitis and 10 to 15 percent brain abscesses. Most commonly, the bacterium is from a lesion outside the central nervous system, typically by infectious disease, either by expansion from parasternal and parameningal abscesses, or by nosocomial infections following neurosurgical procedures. Spread from endocarditis. Over 50% percent of the dural abscess is due to S. aureus; up to half of these cases are associated with purulent spondylitis. Patients have either acute or chronic back pain, usually both fever and fatigue. The onset of nerve root pain is a omen that the disease can progress to neurological dysfunction and eventual paralysis.
[0017]
Antimicrobial resistance due to staphylococcal fever helps maintain them in a hospital environment. Over 90 percent of both S. aureus hospitals and community-type strains that cause infection are resistant to penicillin. This resistance is due to the production of β-lactamase enzymes; the nucleotides encoding these enzymes are usually carried by plasmids. Infections resulting from such acquired resistant microorganisms can sometimes be treated with penicillinase resistant β-lactam antimicrobial agents. However, the true penicillinase-resistant S. aureus bacterium called methicillin-resistant S. aureus (MILSA) is resistant to all of its β-lactam antimicrobial agents as well as the cephalosporin system. MRSA resistance is involved in the production of penicillin binding proteins (PBP 2a and PBP 2 ′) that are mediated on chromosomes and modified with low binding affinity for lactams. MRSA also frequently has acquired plasmid-mediated resistance to erythromycin, tetracycline, chloramphenicol, clindamycin and aminoglycosides. MRSA is becoming increasingly common around the world, especially in specialty care referral hospitals. In the United States, nearly 5 percent of hospital isolates of S. Aureus are methicillin resistant.
[0018]
Thus, there continues to be a need for new agents useful for treating bacterial infections, particularly those caused by antibiotic resistant bacteria, and methods for identifying such new agents. Such a method is ideally independent of the antimicrobial agent present and targets different aspects of host staphylococcal invasion and replication within the host compared to the antimicrobial agent present. To identify the agent.
[0019]
Summary of the Invention
The present invention provides a method of identifying an agent that binds to a polypeptide. The method includes combining the polypeptide and the agent to form a mixture and then determining whether the agent binds to the polypeptide. In some embodiments, the polypeptide may be encoded by a coding sequence having the nucleotide sequence of SEQ ID NO: 7, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, 137 or 141. In another embodiment of the invention, the polypeptide has at least about 57% structural similarity to the nucleotide sequence of SEQ ID NO: 7,21,23,25,27,29,31,33,109,113,117,121,125,129,133 or 137 Cocoded by the essential coding sequence with the sequence. In another embodiment, the polypeptide is encoded by a critical coding sequence having a nucleotide sequence with at least 50% structural similarity to the nucleotide sequence (SEQ ID NO: 141).
[0020]
Determining whether the agent binds to the polypeptide can be accomplished by performing an enzyme assay, a binding assay or a ligand binding assay. The method can further comprise determining whether the agent reduces the growth rate of the microorganism. This involves contacting the microorganism with the agent, incubating the microorganism and the agent under conditions suitable for growth of microorganisms not in contact with the agent, and then determining the growth rate of the microorganism in contact with the agent Including that. A decrease in growth rate compared to a microorganism not in contact with the agent means that the agent decreases the growth rate of the microorganism. The microorganism can be in vitro or in vivo, and the microorganism can be Staphylococcus aureus. The present invention also provides an agent identified by the method.
[0021]
The present invention also provides methods for identifying agents that reduce the growth rate of microorganisms. The method contacts a microorganism with the agent, incubates the microorganism and the agent under conditions suitable for growth of microorganisms not in contact with the agent, and then determines the growth rate of the microorganism in contact with the agent Including doing. A decrease in growth rate compared to a microorganism not in contact with the agent indicates that the agent decreases the growth rate of the microorganism. In some embodiments, the polypeptide may be encoded by a coding sequence having the nucleotide sequence of SEQ ID NOs: 7, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, 137, or 141. In another embodiment of the invention, the polypeptide has at least about 57% structural similarity to the nucleotide sequence of SEQ ID NO: 7,21,23,25,27,29,31,33,109,113,117,121,125,129,133 or 137 It is encoded by an essential coding sequence having a nucleotide sequence. In another embodiment, the polypeptide is encoded by a critical coding sequence having a nucleotide sequence with at least about 57 percent structural similarity to the nucleotide sequence (SEQ ID NO: 141). The microorganism may be in vitro or in vivo, and the microorganism may be Staphylococcus aureus. The present invention also provides an agent identified by the method. The present invention also provides a method for reducing the growth rate of microorganisms. The method includes contacting the microorganism with an agent that binds to the polypeptide. In some embodiments, the polypeptide is encoded by a coding sequence having the nucleotide sequence of SEQ ID NO: 7, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, 137, or 141. In another embodiment of the invention, the polypeptide has at least about 57% structural similarity to the nucleotide sequence of SEQ ID NO: 7,21,23,25,27,29,31,33,109,113,117,121,125,129,133 or 137 Encoded by the nucleotide sequence. In another embodiment, the polypeptide is encoded by a critical coding sequence with at least 57% structural similarity to the nucleotide sequence (141). The microorganism can be in vitro or in vivo, and the microorganism can be Staphylococcus aureus.
[0022]
The present invention provides a method for producing S. aureus with reduced virulence. The method modifies the coding sequence in S. aureus to contain a mutation, and then S. aureus with the mutation has reduced virulence compared to S. aureus without the mutation. Including determining whether to have. A coding sequence modified to include a mutation can comprise the nucleotide sequence of SEQ ID NO: 7, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, 137 or 141. In another embodiment of the invention, the coding sequence modified to contain a mutation is at least about 57 to SEQ ID NO: 7, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133 or 137 nucleotide sequences. An essential coding sequence that can comprise a nucleotide sequence with% structural similarity. In yet another embodiment, the coding sequence modified to contain a mutation can comprise a nucleotide sequence having at least about 57% structural similarity to the nucleotide sequence of SEQ ID NO: 141. Is an array. The present invention also provides S. aureus having reduced virulence and a vaccine composition comprising the S. aureus having reduced virulence.
[0023]
The present invention further provides isolated polynucleotides. The polynucleotide can comprise the nucleotide sequence of SEQ ID NO: 7, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, 137 or 141. In another embodiment, the polynucleotide comprises a nucleotide sequence having at least about 57 percent structural similarity to the nucleotide sequence of SEQ ID NO: 7, 21, 23, 25, 27, 29, 31, 109, 113, 117, 121, 125, 129, 133 or 137. Wherein the isolated polynucleotide comprises an essential coding sequence. In yet another embodiment, the polynucleotide can comprise a nucleotide sequence having at least about 57 percent structural similarity to the nucleotide sequence of SEQ ID NO: 141, wherein the isolated polynucleotide Contains a critical coding sequence. The polynucleotide can also consist essentially of the nucleotide sequence described above, and the polynucleotide can optionally further comprise 0 to about 5,000 nucleotides upstream and / or downstream of the nucleotide sequence. it can. In addition, according to the present invention, a single sequence comprising the amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 110, 114, 118, 122, 126, 130, 134, 138 or 142 is included. A released polypeptide is provided.
[0024]
Definition
As used herein, the term “agent” includes, for example, organic compounds, inorganic compounds, metals, polypeptides, non-ribosomal polypeptides, polyketides, or peptidomimetic compounds that bind to a specific polypeptide. Chemical compound.
[0025]
As used herein, the term “polypeptide” refers to a polymer of amino acids and does not refer to a specific length of a polymer of amino acids. Thus, for example, the terms peptide, oligopeptide, protein and enzyme are included in the definition of polypeptide. The term also includes post-expression modifications of the polypeptide, such as glucosylation, acetylation, phosphorylation, and the like.
[0026]
The term “binds to a polypeptide” refers to the conditions of proximity between an agent and a polypeptide. The association may be non-covalent, where the juxtaposition is energetically favored by hydrogen bonding, van der Waals forces, or electrostatic interactions, or it may be covalent.
[0027]
As used herein, “in vitro” growth of microorganisms refers to, for example, growth in test tubes or agar plates. “In vivo” growth of microorganisms refers, for example, to growth in cultured cells or animals.
As used herein, the terms “microorganism” and “bacteria” are used interchangeably and include unicellular prokaryotic and lower eukaryotic (eg, fungal) organisms, preferably prokaryotes.
[0028]
Detailed Description of the Preferred Embodiments of the Invention
The sequence of the S. aureus genome has been determined and contains approximately 3,500 coding sequences (see, eg, Kunsch et al., EP 0 786 519 A2). As used herein, the terms “coding sequence”, “coding region” and “open reading frame” are used interchangeably herein to encode a polypeptide and under the control of appropriate regulatory sequences. When placed in, refers to a nucleotide sequence that expresses the encoded polypeptide. The boundaries of a coding region are generally determined by a translation stop codon at its 5 'end and a translation stop codon at its 3' end. A regulatory sequence is a nucleotide sequence that regulates the expression of a coding region to which it is operably linked. Non-limiting examples of regulatory sequences include a promoter, transcription start site, translation start site, translation stop site, and terminator site. “Operably linked” refers to a juxtaposition in which components so described are in a relationship permitting them to function as intended. A regulatory sequence is “operably linked” to a coding region when it is ligated in such a way that expression of the coding region is achieved under conditions compatible with the regulatory sequences.
[0029]
At this point, approximately 3,500 coding sequences of the S. aureus genome are not possible to predict some function of the polypeptide predicted to encode. This subset of coding sequences is referred to herein as “unknown coding sequences”. Of the many unknown coding sequences in the S. aureus genome, those required for cell growth are potentially novel targets for antimicrobial therapy. The function of other coding sequences of the S. aureus genome can be hypothesized by comparing the S. aureus coding sequence with a second coding sequence from another organism, where the second coding sequence The sequence has an unknown function. This subset of coding sequences is referred to herein as “known coding sequences”. However, although the function of these coding sequences can be assumed, in many cases it is not known if it is necessary for bacterial growth. Known coding sequences required for bacterial growth are potentially novel targets for antimicrobial therapy.
[0030]
As used herein, a “critical coding sequence” is a bacterial cell, preferably S. epidermidis, S. cerevisiae, in vitro or in vivo, preferably in vitro. It encodes a polypeptide necessary for S. saprophyticus or S. aureus, more preferably S. aureus cells, to grow at a normal growth rate. Such polypeptides are referred to herein as “critical polypeptides”. The coding sequence is such that, at a preferred increase level, mutagenesis of the coding sequence in bacterial cells reduces the growth rate of bacterial cells to less than about 50%, about 60% of the growth rate of bacterial cells not containing the mutated coding sequence. A critical coding sequence when reduced to less than, less than about 80%, most preferably less than about 90%. Methods for measuring the growth rate of microorganisms are well known in the art and are routine. A critical coding sequence can encode a polypeptide with an unknown function, and in some embodiments of the invention encodes a polypeptide with a known function. Preferably the critical coding sequence encodes a polypeptide having an unknown function.
[0031]
Preferably, the critical coding sequence is an essential coding sequence. As used herein, an “essential coding sequence” is a bacterial cell, preferably S. epidermidis, S. saprophyticus, or S. aureus, more preferably S. aureus cells in vitro or in vivo. Preferably, it is a coding sequence that encodes a polypeptide that is essential for growth in vitro. Such polypeptides are referred to herein as “essential polypeptides”. The essential coding sequence can encode a polypeptide having an unknown function, or in some embodiments of the invention, encodes a polypeptide having a known function. Preferably, the essential coding sequence encodes an unknown functional polypeptide.
[0032]
The identification of these critical coding sequences, preferably essential coding sequences, can be used to inhibit bacteria, preferably S. epidermidis, S. saprophyticus or S. aureus, more preferably S. aureus. Compared to agents, it provides a means to discover new agents with different targets and mechanisms of action. Identification of the essential coding sequence of a microorganism useful in the present invention, preferably S. epidermidis, S. saprophyticus or S. aureus, more preferably S. aureus, is a polypeptide, preferably a polymorphism having no known function. One can start by identifying the coding sequence that encodes the peptide. Coding sequences can be located in databases including, for example, the University of Oklahoma, TIGR, NCBI, Sanger, HGS Contig database, and S. Aureus database available from the HGS GSTS database. Identification of such a coding sequence involves constructing a contig from data present in such a database.
[0033]
As described herein, unknown coding sequences were typically identified by analyzing publicly known polynucleotide sequences. The data obtained from the database included genomic clones and predicted open reading frame nucleotide sequences. However, even though the putative coding sequence was known, there was no indication that the coding sequence was in fact expressed or in fact a critical coding sequence. For example, the data known to those skilled in the art are limited regarding the regulatory regions required for transcription of nucleotide sequences in S. aureus. Furthermore, there is generally no evidence that the critical and essential coding sequences identified herein are actually expressed. Thus, one of ordinary skill in the art having a polynucleotide sequence of a genomic clone would not be able to predict that an open reading frame will be transcribed or that the coding sequence is critical, preferably essential.
[0034]
Typically, whether a coding sequence is a critical coding sequence, preferably an essential coding sequence, is determined by inactivating the coding sequence in the bacterial cell and then determining the growth rate of the bacterial cell. Can do. Proliferation can be measured in vitro or in vivo, preferably in vitro. Inactivation of the coding sequence is done by mutating the coding sequence present in the bacterial cell. Mutations include, for example, deletion mutations (ie, deletion of nucleotides from the coding sequence), insertion mutations (ie, insertion of additional nucleotides into the coding sequence), nonsense mutations (ie, changes in codon nucleotides). Thus, the codon encodes a different amino acid), and missense mutations (ie, changes in the nucleotides of the codon, and thus the codon functions as a stop codon). Some insertion and deletion mutations result in shift mutations. Preferably, the coding sequence in the bacterial cell is made to contain a deletion.
[0035]
In general, internal fragments of a selected coding sequence can be isolated or synthesized by methods known in the art including, for example, polymerase chain reaction (PCR). Typically, the internal fragment is about 150 base pairs, about 350 base pairs, preferably about 300 base pairs in length. The internal fragment corresponds to the 5 'end of this coding sequence. Preferably, the primer used to amplify the internal fragment contains a restriction site that allows the amplified internal fragment to be linked to a vector. For example. When the vector is pSPT246 (described below), one primer can contain a PstI site and the other primer can contain a SacI site.
[0036]
The internal fragment is typically linked to a vector, which can be used to inactivate the coding sequence in bacterial cells and determine whether the coding sequence is a critical coding sequence or an essential coding sequence. can do. Useful vectors include those that cannot replicate under certain conditions in bacterial cells containing the coding sequence to be inactivated. Preferably, the vector is a temperature sensitive vector, i.e. it cannot replicate with S. aureus at elevated temperatures, e.g. at least about 42 <0> C, or the vector is a suicide vector, i.e. it is S. aureus. Can not be duplicated. Preferably, the temperature sensitive vector is a shuttle vector, i.e. capable of replication in E. coli and S. aureus under suitable conditions. Examples of temperature sensitive plasmids that can be used to inactivate coding sequences in S. aureus include pSPT181 (Janzon and Arvidson, EMBO J., 9, 1391-1399 (1990)), and pSPT246 (discussed below). . Examples of suicide plasmids that can be used to inactivate coding sequences in S. aureus include pKT4 (Tegmark et al., Mol. Microbiol., 37, 398-409 (2000)).
[0037]
Using these methods, the following essential coding sequences were identified: SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 33, 109, 113, 117, 121, 125, 129, 133 and 137. The polypeptides encoded by the coding sequences are SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 110, 114, 118, 122, 126, 130, 134, and 138. Of these essential coding sequences, one coding sequence (SEQ ID NO: 33) encodes uridylate kinase, and thus prior to the present invention, which is a known coding sequence, the uridylate kinase coding sequence is S. aureus. It was not known to be essential for the growth of Using these methods, a critical coding sequence having the DNA sequence set forth in SEQ ID NO: 141 was identified. The polypeptide encoded by the critical coding sequence is SEQ ID NO: 142.
[0038]
The coding sequence of the present invention is similar to the coding sequence present in SEQ ID NO: 1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,109,113,117,121,125,129,133,137,141 Contains the coding sequence or its complement. Similarity refers to structural similarity between two polynucleotide residues (ie, candidate coding sequences and SEQ ID NOs: 1,3,5,7,9,11,13,15,17,19,21, Determined by aligning the nucleotide sequences of the coding regions of 23,25,27,29,31,33,109,113,117,121,125,129,133,137,141, or their complements, to optimize the number of identical nucleotides along their length Gaps in either or both of the sequences are allowed in making an alignment that optimizes the number of shared nucleotides, but the nucleotides in each sequence must remain in their proper order. Candidate coding sequences are coding regions present in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, 137 or 141, or A coding region to be compared with its complement. Candidate nucleotide sequences can be isolated from microorganisms, preferably S. aureus, or can be produced using recombinant techniques, or can be synthesized chemically or enzymatically. Preferably, the Blast 2 program of the BLAST 2 search algorithm is described as described by Tatusova et al. (FEMS Microbiol Lett 1999, 174: 247-250) and available at www.ncbi.nlm.nih.gov/gorf/bl2.html. To be compared. Preferably, all points including score for match = 1, penalty for mismatch = -2, open gap penalty = 5, extended gap penalty = 2, gap x dropoff = 50, prediction = 10, word length = 11, etc. Use defect values for BLAST2 search parameters. In a comparison of two nucleotide sequences using the BLAST search algorithm, structural similarity refers to “identity”. Preferably, the polynucleotide is against the coding region of SEQ ID NO: 1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,109,113,117,121,125,129,133,137,141 or its complement At least about 57%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, and most preferably at least about 95% in order of increasing preference. Contains nucleotide sequences of identity.
[0039]
The present invention provides an isolation comprising the nucleotide sequence SEQ ID NO: 1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,109,113,117,121,125,129,133,137,141 or its complement A modified polynucleotide. As used herein, an “isolated” polypeptide or polynucleotide has been removed from its natural environment, produced using recombinant techniques, or synthesized chemically or enzymatically. Means polypeptide or polynucleotide. Preferably, the polypeptide or polynucleotide of the invention is purified, i.e. substantially free of any other polypeptide or polynucleotide and associated cell products or other impurities. The isolated polypeptide of the present invention has the nucleotide sequence SEQ ID NO: 1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,109,113,117,121,125,129,133,137,141 Nucleotide sequences having a structural similarity of at least about 57%, at least about 60, at least about 70%, at least about 80, at least about 90%, most preferably at least about 95% in order of increasing preference for The isolated polynucleotide may comprise a critical coding sequence, preferably an essential coding sequence. The invention also includes a polypeptide encoded by the coding sequence.
[0040]
Another aspect of the present invention is the nucleotide sequence SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, 137, or 141 Or an isolated polynucleotide consisting essentially of its complement. The polynucleotide may further comprise a nucleotide sequence SEQ ID NO: 1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,109,113,117,121,125,129,133,137,141 or Contains 0 to 5,000 nucleotides upstream and / or downstream of its complement. The isolated polynucleotide of the present invention has the nucleotide sequence SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, 137, 141 or At least about 57%, at least about 60%, at least about 70%, at least 80%, at least 90%, most preferably at least about 95% structural similarity in order of increasing preference for its complement The isolated polynucleotide can comprise an essential coding sequence, wherein the isolated polynucleotide comprises an essential coding sequence. The polynucleotide may further comprise a nucleotide sequence SEQ ID NO: 1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,109,113,117,121,125,129,133,137,141 or Contains 0 to 5,000 nucleotides upstream and / or downstream of its complement. The invention also includes a polypeptide encoded by the coding sequence.
[0041]
Insertional inactivation of critical coding sequences, preferably essential coding sequences, allows different classes of coding sequences to be identified. Examples of different classes include, for example, coding sequences encoding proteins involved in cell surface metabolism, enzymes involved in cell biosynthetic pathways including cell wall biosynthesis and assembly, components of the TCA circuit, ATP-binding cassette (ABC A) Similar to the transporter superfamily of oligopeptide transport proteins and includes proteins involved in cellular regulation and repair processes, and coding sequences that affect morphology and cell division, protein secretion and classification, and signal transduction systems.
[0042]
The critical coding sequence, preferably the essential coding sequence, can be cloned by PCR using microorganisms, preferably S. epidermidis, S. saprophyticus or S. aureus, more preferably S. aureus genomic DNA as template. . For ease of insertion into the open reading frame, the PCR primers consist of a 5 'end restriction enzyme whose PCR-amplified coding sequence precedes the start codon ATG and a 3' end restriction enzyme after the stop codon TAG, TGA or TAA Can be selected. If desired, the codons in the coding sequence can be changed without changing the amino acid to optimize expression of the polypeptide encoded by the essential coding sequence. For example, if the essential coding sequence is to be expressed in E. coli, the codon of the coding sequence can be changed to match the preference of the E. coli codon (eg, Grosjean and Fiers, Gene, 18 , 199-209 (1982), and Konigsberg et al., Proc. Natl. Acad. Sci., USA, 80, 687-691 (1983)). Optimization of codon usage can lead to increased expression of the encoded polypeptide when the essential coding sequence is produced in a microorganism other than the microorganism from which it was isolated. If the polypeptide is to be produced extracellularly, e.g., in E. coli or other bacterial periplasm, or in cell culture medium, the coding sequence is cloned without its start codon and expressed in an expression vector after the signal sequence. Can be put.
[0043]
Proteins can be produced in prokaryotic or eukaryotic expression systems using known promoters, vectors and hosts. Such expression systems, promoters, vectors and hosts are known in the art. Such as other bacteria including E. coli, Bacillus and S. aureus, yeasts including Pichia pastoris and Saccharomyces cerevisiae, insect cells, or mammalian cells including SHO cells Appropriate host cells can be used to express the polypeptide using any suitable vector known in the art. Proteins can be produced directly in the cell or extracellularly by secretion into the periplasmic space or cell culture medium of bacterial cells, or can be fused to polypeptides. Protein secretion typically requires a signal peptide (also known as a pre-sequence); many signal sequences from prokaryotes and eukaryotes function in the secretion of recombinant proteins. It has been known. During the protein secretion process, the signal peptide is removed by signal peptidase to obtain the mature protein.
[0044]
A polypeptide encoded by a critical coding sequence, preferably an essential coding sequence, can be isolated. A purification tag can be added to either the 5 ′ or 3 ′ end of the coding sequence to simplify the isolation process. Commonly used purification tags are three stretches of histidine residues (US Pat. Nos. 5,284,933 and 5,310,663), Schmidt and Skerra, Protein Engineering, 6, 109-122 (1993). Streptavidin-affinity tag, FLAG peptide (Hopp et al., Biotechnology, 6, 1205-1210 (1988)), glutathione S-transferase (Smith and Johnson, Gene, 67, 31-40 (1988)) And thioredoxin (LaVallie et al., Bio / Technology, 11, 187-193 (1993)). To remove these tags, proteolytic cleavage recognition sites can be inserted into the fusion junction. Commonly used proteases are factor Xa, thrombin and enterokinase. Preferably, the polypeptide encoded by the essential coding sequence is isolated and more preferably purified.
[0045]
The identification of critical coding sequences, preferably essential coding sequences, makes them useful in the method of identifying new agents according to the present invention. Such methods assay potential agents for the ability to interfere with expression of critical coding sequences, preferably essential coding sequences, thereby preventing expression of the polypeptide encoded by the coding sequence and reducing its concentration. Including doing. Without intending to be limiting, the agent interacts with, for example, a critical coding sequence, preferably an essential coding sequence, and interacts with a nucleotide sequence adjacent to the critical coding sequence, preferably an essential coding sequence (eg, a promoter sequence). It is expected that it can act by inhibiting the expression of a polypeptide involved in the regulation or expression of a critical coding sequence, preferably the essential coding region. Agents that can be used to inhibit expression of critical coding sequences, preferably essential coding regions, include, for example, antisense polypeptides complementary to mRNA molecules transcribed from coding sequences, and the use of double-stranded RNA (Fire et al., Nature, 391,806-11 (1998)).
[0046]
Such a method is described in any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, 137, 141. Assaying potential agents for the ability to bind to a polypeptide that is wholly or partially encoded by a DNA sequence or its complementary strand. If desired, agents that bind to such polypeptides can be further evaluated by determining if they inhibit the function of the polypeptide to which they bind.
[0047]
Polypeptides produced by a critical coding sequence, preferably an essential coding sequence, can be used in assays, including, for example, high-throughput assays, to screen for agents that inhibit the function of the polypeptide. Sources for potential agents to be screened include, for example, chemical compound libraries, Streptomysetes, other bacterial and fungal fermentation media, and plant and other vegetative cell extracts. For proteins with known enzymatic activity, assays can be established based on activity and a large number of potential agents can be screened for the ability to inhibit activity. Such assays are referred to herein as “enzyme assays”. Enzymatic assays vary depending on the enzyme and are typically known in the art.
[0048]
For proteins that interact with another protein or nucleic acid, an assay can be established to directly measure such interactions, and potential agents are screened for the ability to inhibit binding interactions. (Referred to herein as a “binding assay”). In another aspect of the invention, an assay can be established to allow identification of an agent that binds to a polypeptide encoded by an essential coding sequence (referred to herein as a “ligand binding assay”). .
[0049]
For proteins that interact with another protein or nucleic acid, such binding interactions are described in Fields and Song (Nature, 340, 245-246 (1989)) and Fields and Sternglanz (Trends in Genetics, 10, 286-292 ( 1994)) can be used for indirect evaluation. The two-hybrid system is a genetic assay for detecting an interaction between two polypeptides. It can be used to identify proteins that bind to known proteins of interest, or to represent domains or residues that are critical for interaction. Variations on this method are developed to clone the coding sequence encoding the DNA-binding protein, identify polypeptides that bind to the protein, and screen for drugs. The two-hybrid system has developed the ability of a pair of interacting proteins to bring the transcriptional activation domain close to the DNA-binding domain that binds to the upstream activation sequence (UAS) of the reporter coding sequence, which is generally yeast. Done in The assay requires the construction of a two-hybrid coding sequence encoding (1) a DNA-binding domain fused to protein X and (2) an activation domain fused to protein Y. The DNA-binding domain targets the first hybrid protein to the reporter coding sequence UAS; however, since most proteins lack the activation domain, this DNA-binding hybrid protein activates transcription of the reporter coding sequence. Do not turn. The second hybrid protein containing the activation domain cannot itself activate reporter expression. Because it doesn't bind to UAS. However, in the presence of both hybrid proteins, the non-covalent interaction of protein X and protein Y links the activation domain to the UAS and activates transcription of the reporter coding sequence. For example, if it is already known that a polypeptide encoded by an essential coding sequence (eg, protein X) already interacts with another protein or nucleic acid (eg, protein Y), this binding assay can be used to Agents that interfere with the Y and Y interactions can be detected. The expression of the reporter coding sequence is monitored as different test agents are added to the system; the presence of the inhibitor inhibits binding and consequently the reporter signal is absent.
[0050]
For example, if the function of the polypeptide encoded by the essential coding sequence is unknown and the ligand that binds to the polypeptide is not known, the yeast two-hybrid assay is also used to identify the protein that binds to the polypeptide. can do. In assays to identify proteins that bind to protein X (target proteins), a large number of hybrid coding sequences, each containing a different protein Y, are produced and screened in the assay. Typically, Y is encoded by a pool of plasmids in which whole cDNA or genomic DNA is linked to the activation domain. This system can be applied to a wide variety of proteins and does not even need to know the identity or function of protein Y. The system is quite sensitive and can detect interactions that have not been revealed by other methods; even transient interactions can trigger transcription to produce stable mRNAs that can be translated repeatedly; A reporter protein is obtained. Once a protein that binds to the essential polypeptide is identified, the two-hybrid system can be used in a binding assay to identify agents that inhibit binding and consequently lack a reporter signal.
[0051]
One can search for agents that bind to the target protein using ligand binding assays known in the art. Without intending to be limiting, one such screening method for identifying direct binding of a test ligand to a target protein is described in Bowie et al. (US Pat. No. 5,585,277). This method relies on the principle that proteins are generally present as a mixture of folded and unfolded states and change continuously between the two states. When the test ligand binds to the folded form of the target protein (ie, when the test ligand is a ligand of the target protein), the target protein molecule to which the ligand is bound is still in its folded state. Thus, the folded target protein is present to a greater extent in the presence of a test ligand that binds to the target protein than in the absence of the ligand. Ligand binding to the target protein can be measured by any method that distinguishes between the folded and unfolded states of the target protein. The function of the target protein need not be known to perform this assay.
[0052]
Another method for identifying ligands for target proteins is described in Wieboldt et al., Anal. Chem., 69, 1683-1691 (1997). This technique screens a combinatorial library of 20-30 agents at a time in liquid phase for binding to the target protein. Agents that bind to the target protein are separated from other library components by centrifugal ultrafiltration. The specifically selected molecules retained on the filter are subsequently released from the target protein and analyzed by HPLC and hydrodynamic assisted electron spray (ion spray) ionization mass spectrometry. This approach selects library components with greater affinity for the target protein and is particularly useful with small molecule libraries.
[0053]
Another method allows the identification of ligands present in a sample using capillary electrophoresis (Hughes et al., US Pat. No. 5,783,397). Combine sample and target protein and degrade. As a result of the electrophoresis conditions, the components present in the sample are fractionated simultaneously and screened for components that bind to the target molecule. This method is particularly useful for complex samples including, for example, extracts of plants, animals, microorganisms, or parts thereof, and chemical libraries produced, for example, by combinatorial chemistry.
[0054]
Agents identified by initial screening are evaluated for their effects on the survival of microorganisms, preferably S. epidermidis, S. saprophyticus, S. aureus, more preferably S. aureus. Agents that interfere with bacterial survival are expected to prevent the establishment of infection or, once it is established, can reverse the outcome of the infection. The agent can be bactericidal (ie, an agent that kills microorganisms and prevents microbial replication) or bacteriostatic (ie, an agent that reversibly prevents microbial replication).
[0055]
Preferably the agent is bactericidal. Such an agent is infected with S. epidermidis, S. saprophyticus or S. aureus, preferably S. aureus, or S. epidermidis, S. saprophyticus or S. aureus, preferably S. aureus. It may be useful to treat subjects at risk of being infected with Aureus.
[0056]
The identification of S. aureus critical coding sequences, preferably essential coding sequences, also provides microorganisms exhibiting reduced virulence useful in vaccines. The term “vaccine” refers to a composition that provides protection against S. epidermidis, S. saprophyticus or S. aureus, preferably S. aureus, upon administration to a subject. Administration of the vaccine to the subject produces an immunological response to S. aureus, resulting in immunity. Vaccines are effective in providing some therapeutic benefit or effect to produce an immune response that inhibits or prevents infection with S. aureus in a subject or to produce antibodies against S. aureus Is administered in appropriate amounts.
[0057]
Such microorganisms that can be used in the vaccine are any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, 137, 141 S. epidermidis, S. saprophyticus or S. aureus, preferably S. aureus mutants containing mutations in the coding sequence represented by or one of the coding sequences having structural similarity thereto Including. If desired, S. epidermidis, S. saprophyticus or S. aureus, preferably S. aureus, contains more than one mutation. Reduced virulence of these organisms and their immunogenicity can be confirmed by administration to a subject. Animal models useful for assessing S. aureus virulence in various diseases including, for example, pneumonia, peritonitis, endophthalmitis, endocarditis, sepsis and arthritis are known in the art.
[0058]
Where the avirulent microorganism of the invention can be administered alone, one or more such mutant microorganisms preferably contain a suitable adjuvant (s) and a pharmaceutically acceptable diluent (s) or carrier (s). To be administered in a vaccine composition. The carrier (s) must be “acceptable” in the sense of being compatible with the avirulent microorganism of the present invention and not harmful to the subject to be immunized. Typically, the carrier will be sterile or pyrogen-free water or saline. The subject to be immunized is a subject in need of protection from illness caused by S. aureus virulent form.
[0059]
Oil-based adjuvants, such as Freund's complete and incomplete adjuvants, mycolate-based adjuvants (eg trehalose dimycolate), bacterial lipopolysaccharide (LPS), peptidoglycans (ie mumine, mucopeptide or N-Opaca Glycoproteins, muramyl dipeptides (MDP or MDP analogs), proteoglycans (eg extracted from Klebsiela spp), streptococcal preparations (eg OK 432), EP 109 942, EP 180 564 and EP231 039 “Iscoms ”, Aluminum hydroxide, saponin, DEAE-dextran, neutral oil (such as miglyol), vegetable oil such as peanut oil, liposomes, Ribi adjuvant system (see eg GB-A-2 189 141) or BIOSTIM (eg ) And Pluronic poly Vaccine compositions of any adjuvant known in the art, including oals, can be used in recent days.Another adjuvant consisting of an extract of Amycolata, a bacterial genus in the order Actinomycetales, has been described in US Pat. In addition, registered trade name adjuvant mixtures are commercially available, the adjuvant used depends in part on the recipient organism, and the amount of adjuvant to be administered depends on the type and size of the animal. Optimal dosages can be readily determined by routine methods.
[0060]
The vaccine composition can optionally include a pharmaceutically acceptable (ie, sterile and non-toxic) liquid, semi-solid or solid diluent that acts as a pharmaceutical vehicle, excipient or vehicle. Any diluent known in the art can be used. Examples of diluents include, but are not limited to, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl- and propylhydroxybenzoate, talc, alginate, starch, lactose, sucrose, dextrose, sorbitol, mannitol, acacia Includes gum, calcium phosphate, mineral oil, cocoa butter and theobroma oil.
[0061]
The vaccine composition can be packaged in a form convenient for delivery. The composition can be contained in capsules, sachets, cachets, gelatin, paper or other containers. These delivery forms are preferred when suitable for entry of the immunogenic composition into the recipient organism, particularly when the immunogenic composition is delivered in a unit dosage form. Dosage units can be packaged, for example, in tablets, capsules, suppositories, or cachets. The vaccine composition includes, for example, by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, or subcutaneous injection: oral, sublingual, nasal, anal, vaginal, transdermal and / or by surgical implantation. Any conventional method can be introduced into a subject to be immunized, for example, embedded under a spleen capsule or in the cornea. The treatment can consist of a single dose or multiple doses over time.
[0062]
It will be appreciated that the vaccines of the present invention are useful in the fields of human medicine and veterinary medicine. Thus, the subject to be immunized can be a human or animal such as cattle, sheep, pigs, horses, dogs, cats, chickens such as chickens, turkeys, ducks and geese, and the following examples illustrate the invention. It should be understood that specific examples, materials, amounts and techniques should be broadly construed in accordance with the scope and spirit of the invention described herein.
[0063]
Example 1
Identification of critical and essential S. aureus coding sequences
Identification of unknown coding sequences
There are about 3500 open reading frames in the HGS database of S. aureus nucleotide sequences. A fast A homology search was performed on these open reading frames. A search for these open reading frames indicated that 662 of the open reading frames were unknown coding sequences. The methods described herein typically require an open reading frame of about 100 base pairs; 492 of the 662 open reading frames were at least 300 base pairs. Of these 492, 60 have homology to unknown open reading frames from other bacteria, 270 have no homology to any open reading frame, and 160 are eukaryotic Has homology to the coding sequence. A homology search was also performed between the predicted coding sequence of the Mycoplasma genitalium genome and the coding sequence of S. aureus. The nucleotide sequence of the unknown coding sequence is shown in Table 1. These reading sequences were determined as critical or essential as described herein.
[0064]
[Table 1]

[0065]
Insertion inactivation of unknown coding sequence
Inactivation was achieved by integrating the plasmid into the 5 'half of the target coding sequence by homologous recombination. An internal fragment of the selected coding sequence was synthesized by PCR. The length of the amplified fragment was between about 250 base pairs to about 350 base pairs and included the 5 'end of the coding sequence. The primers used in the amplification included additional nucleotides to add one end of the PstI restriction site amplified fragment and a SacI restriction site to the other end of the amplified fragment. The primers are shown in Table 1. The added restriction site allowed the amplified fragment to be ligated to the temperature sensitive shuttle vector pSPT246. pSPT264 was constructed by ligating pRN8103 and pSP64-PolyA. The pRN8103 temperature sensitive replication vector contains a unique EcoRI restriction site, which cannot replicate in E. coli. pRN8103 is described in Novick et al. (J. Mol. Biol., 192, 209-220 (1986)). The pSP64-PolyA vector obtained from Promega Corp. (Mabison, Wis.) Replicates in E. coli but not in S. aureus. pSP64-PolyA also contains a unique EcoRI restriction site. The E. coli / S. Aureus shuttle vector was constructed by digesting each vector with EcoRI, ligating the two vectors together, and then transforming the DNA into E. coli. The obtained shuttle vector was named pSPT264.
[0066]
A recombinant plasmid (ie, pSPT246 containing the amplified fragment) is used to transform E. coli and then isolated by electroporation (Kreiswirth et al., Nature, 305, 709-712 (1983) Transferred to S. Aureus RN4220). Transformants were selected by incubating at an acceptable temperature (30 ° C.) on nutrient agar plates containing tetracycline (10 μg / ml). The presence of the correct plasmid was confirmed by PCR.
[0067]
One clone with the correct plasmid was grown overnight at 32 ° C. on nutrient agar containing tetracycline (10 μg / ml) to effect recombination between the plasmid and the selected chromosomal allele. To select for recombinants, the bacteria were then grown for 18 hours at an unacceptable temperature (43 ° C.) in brain heart infusion (BHI) broth without tetracycline, followed by 5 μg / Dilute 1:10 in BHI broth containing ml tetracycline. Cells were incubated overnight at 43 ° C. The bacterial culture was then diluted and spread on nutrient agar plates containing 5 μg / ml tetracycline and incubated overnight at 43 ° C. Since the plasmid cannot replicate at 43 ° C., only cells with the plasmid integrated into the chromosome are tetracycline resistant and form colonies. Also considered are microcolonies that appear at unacceptable temperatures. This is because they can show mutations in coding sequences that are important (eg critical) but not essential for growth.
[0068]
The plasmid is less integrated at other sites in the chromosome, and thus a tetracycline resistant clone appeared only when the target coding sequence was essential. Thus, 10 colonies from each selection at 43 ° C. were tested for specific integration of the plasmid into the selected target coding sequence by PCR. A primer pair consisting of one primer that binds to the vector DNA and a second primer that binds upstream of the target coding sequence in the chromosome was used for PCR amplification. The primer pair amplifies the intervening chromosome-vector region and an amplified DNA fragment is produced only if the vector is integrated at the expected position. The absence of bands suggests that the vector cannot be integrated and that the coding sequence is essential. Typically, all of the tested colonies were specific recombinants or none of the recombinants. If no recombinant is found, the target coding sequence is considered essential. For a large number of target coding sequences (both essential and non-essential), identical results were obtained when the entire selection procedure was repeated.
[0069]
This protocol has been successfully used to analyze approximately 300 of the 492 unknown complete or partial coding sequences identified. Of the coding sequences analyzed, 26 seemed critical and were further analyzed as described below.
[0070]
Example 2
Cloning of essential S. aureus coding sequence and expression in E. coli
Overview of expression systems and cloning techniques
Overexpression of S. aureus protein is achieved using the Qiagen type ATG expression system (Qiagen Gmbh, Santa Clara, CA). This system has a genotype of Nal ^s , Str ^s , Rif ^s , Lac ^- , Ara ^- , Gal ^- , Mtl ^- , F ^- , RecA ⁺ , Uvr ⁺ E. coli strain “M15” described by Qiagen as Two replication compatible vectors, pREP4 and pQE-60 (each obtained from Qiagen) are introduced into the M15 strain during the procedure. Alternatively, pQE-70 can be used in place of pQE-60. The pREP4 vector is a pACYC-derived vector containing the lacI gene encoding for the lactose (LacI) repressor protein, which encodes kanamycin drug resistance. The expression vector pQE-60 is a pBR322-derived vector containing a modified T5 phage promoter, a strong ribosome binding site (RBS), and the coding sequence of the specific S. aureus coding sequence to be expressed. Modification of the T5 promoter involves replacement of the operator for binding and regulation of the promoter by the LacI repressor. Induction of expression is performed by adding IPTG (isopropylthio-β-D-galactoside) to log phase culture.
[0071]
A common cloning strategy is to first amplify specific coding sequences from S. aureus genomic DNA using PCR primers for the 5 'and 3' ends of the coding sequence. The PCR primers are designed to add NcoI and BglII restriction sites at the 5 ′ and 3 ′ ends of the coding sequence, respectively. The coding sequence should not contain any NcoI or BglII restriction sites. If such a site is present, it is eliminated using site-specific PCR mutagenesis techniques known to those skilled in the art. Alternatively, a different restriction site, such as a BamHI restriction site, is used in place of the BglII restriction site. The amplified S. aureus coding sequence is ligated into pCR-2.1 (Invitrogen, Carlsbad, Calif.) And transformed into E. coli using techniques known in the art. Colonies are screened for the presence of the coding sequence by PCR amplification or vector restriction analysis. Clones are selected randomly and the nucleotide sequence of the inserted DNA, ie, the S. aureus coding sequence, is measured to confirm the authenticity of the insert.
[0072]
The pCR-2.1 vector containing the desired coding sequence is digested with NcoI / BglII, the coding sequence is isolated and ligated to the corresponding NcoI / BglII restriction sites of pQE-60. Using the ligation mixture, vector DNA is electroporated into the M15 strain containing the pREP4 vector. The resulting transformants are screened by PCR or restriction analysis. Candidates are grown in shake flasks and analyzed by SDS-PAGE or Western analysis to screen for overexpression of protein bands with the appropriate size. Anti-His antibody (Invitrogen) is used for Western analysis. A single candidate is selected for overexpression and isolation of the protein encoded by each coding sequence.
[0073]
Culture and media
The medium for cloning and maintenance of cells containing recombinant plasmids in E. coli is LB supplemented with appropriate antibiotics (100 μg / ml ampicillin, 25 μg / ml kanamycin). S. aureus was grown in Mueller Hinton medium. Competent INVF'α cells (Invitrogen, Carlsbad, CA) are used according to the manufacturer's instructions. M15 pREP-4 strain was purchased from Qiagen. For electroporation of cells, SOC medium was used. LB and SOC media are described in Sambrook et al. (Molecular Cloning: A Laboratory Manual., Cold Spring Harbor Laboratory Press, pp. Al-A4 (1989)). Mueller-Hinton medium is described in Atlas et al., Handbook of Microbiological Media, CRC Press.
[0074]
pQE60 expression vector design
PQE-60 DNA sequence, including T5 promoter, RBS, ATG start codon (bold), NcoI restriction site (underlined), BglII restriction site (underlined), 6His tag (double underline), and TAA stop codon (bold) A part is shown (SEQ ID NO: 89):
[0075]
[0076]
[Chemical 1]

[0077]
The S. aureus coding sequence is modified by PCR to include compatible in-frame NcoI and BglII restriction sites.
[0078]
Primer design
The general formula for primer design for the 5 'part of the S. aureus coding sequence is usually 5'-CCATGGGAN _20-30 The general formula for the 3 'primer is usually 5'-AGATCTN _20-30 It is. These primers add NcoI and BglII restriction sequences. The first “N” nucleotide of the 5 ′ sequence corresponds to the codon of the second amino acid of the S. aureus coding sequence after the start of its ATG. The first “N” nucleotide of the 3 ′ primer corresponds to the third nucleotide in the codon preceding the stop codon of the S. aureus coding sequence. The number of nucleotides contained in the primer varies depending on the specific DNA sequence, but typically ranges from 20 to 30 bases. The primer is phosphorylated. Examples of primers that can be used to amplify several coding sequences are shown in Table 2.
[0079]
[Table 2]

[0080]
Preparation of S. aureus genomic DNA
Strain ISP3 (obtained from S. Arvidson, Karolinska Institute) is used to inoculate 10 ml of Mueller Hinton broth. After overnight growth at 37 ° C., 1.5 ml cultures are pelleted in Eppendorf tubes and then resuspended in 400 μl TE (pH 8.0) (Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, p. B.20 (1989)) Following the addition of 50 μl lysostaphin solution (10 mg / ml), the cells are incubated for 1 hour at 37 ° C. 70 μl 10% SDS and 10 μl Proteinase K (20 mg / ml) is added and incubation is continued for an additional hour at 37 ° C. After addition of 100 μl 5 mL NaCl, the cell suspension is stirred and 10% hexadecyltrimethylammonium bromide, 0%. Add 80 μl of a solution containing 7 M NaCl (CTAB / NaCl) Stir cells then incubate for 10 minutes at 65 ° C. Equal volume of 25: Following the addition of 24: 1 phenol: chloroform: isoamyl alcohol, the cells are agitated and centrifuged for 5 minutes, and then the aqueous layer is transferred to a fresh tube to obtain a white CTAB / NaCl interface. Transfer the layer back to a fresh test tube, followed by addition of an equal volume of isopropanol, mix the test tube gently until a filamentous precipitate is formed, and use a small hook-shaped Pasteur pipette to remove the precipitate. Remove gently and transfer it to another tube containing 1 ml of 70% ethanol, centrifuge the tube and wash the resulting pellet once with 70% ethanol. The pellet is resuspended in 100 μl water and the concentration of recovered DNA is measured using techniques known in the art.
[0081]
PCR amplification
PCR reactions are performed using either a Perkin-Elmer Cetus GeneAmp 9600 or 2400 thermal cycler (Perkin-Elmer, Norwalk, CT). Deoxynucleotide mix and Pfu DNA polymerase are purchased from Stratagene (La Jolla, CA). The AmpliTaq Gold kit is purchased from Perkin Elmer. The PCR synthesis protocol for long template amplification is as follows: 1 μg S. aureus genomic DNA, 10 μl 10 × reaction buffer (15 mm MgCl 2 ₂ ), 500 mg of each primer, 16 μl of 1.25 mM dNTP, 1 μl of AmpliTaq Gold, and up to 100 μl of water are added per PCR microtube. Using a cycle program of 95 ° C for 5 minutes followed by 35 cycles of 94 ° C for 30 seconds, 50 ° C for 1 minute and 72 ° C for 3 minutes, extension at 72 ° C for 5 minutes, and last 40 ° C hold 35 Amplify DNA in a cycle. A 10 μl aliquot of the synthesis reaction is loaded onto a 1.2% agarose gel to confirm the presence and size of the synthesized fragment. PCR products are generated by combining multiplex PCR reactions, precipitating DNA with EtOH, and then cleaving the desired fragment from a 1.2% agarose gel. DNA is isolated from agarose using an Amicon Ultrafree-DA extraction filter (Millipore Corp., Bedford, Mass.). Use the filter according to the manufacturer's instructions.
[0082]
Ligation and transformation
The pQE-60 vector and the pCR2.1 vector containing the S. aureus coding sequence are digested with NcoI and BglII restriction enzymes. The pQE-60 vector fragment and the S. aureus coding sequence are isolated from the agarose gel. The two DNAs were ligated and transformed into electrocompetent M15 cells containing pREP-4, supplemented with ampicillin and kanamycin. Plate on LD agar. Ligase is purchased from BioLab (Beverley, MA) and used according to the manufacturer's instructions. Electroporation of the ligated DNA into M15 pREP-4 cells is performed using a Bio-Rad Gene pulser (Hercules, CA). A ₅₅₀ Competent cells are prepared from 1 liter of cells having an optical density of 1. Cells are cooled and washed sequentially with 1 liter and 0.5 liter ice cold sterile water. Cells are resuspended in 20 ml ice cold sterile glycerol, centrifuged again and placed in a final suspension of 2-3 ml cold sterile 10% glycerol. 50 μl of cells are mixed with 5 μl or less of ligated DNA. The cell / DNA mixture is transferred to an electroporation cuvette and pulsed with a pulse controller set at 25 μF, 2.5 kV, and 200Ω. Then 1 ml of SOC medium is added. Cells are incubated at 30 ° C. and placed on selective medium.
[0083]
Several resulting colonies from the transformation are randomly selected and vector DNA is isolated using a Miniprep or Maxiprep kit purchased from Qiagen. Vector DNA is isolated according to the manufacturer's instructions. Candidates are screened by restriction enzyme digestion. Restriction enzymes are purchased from New England BioLab (Beverly, MA). Restriction enzymes are used according to the manufacturer's instructions.
[0084]
Expression conditions
Expression cultures are streaked on LB plates containing ampicillin and kanamycin. Single colony isolates are used to inoculate 50 ml of LB medium supplemented with ampicillin and kanamycin and grown overnight at the desired temperature. 0.50A ₅₅₀ Subculture to the same volume of the same medium at / ml, followed by 3.0 A ₅₅₀ The culture is grown with vigorous aeration at the same temperature until reaching The culture is induced to a final concentration of 1 mM by addition of IPTG. Culture aliquots are removed at 0, 2 and 4 hours after induction for SDS-PAGE or Western analysis. Cells are harvested for protein isolation between 4 and 6 hours. Proteins are isolated using a metal chelate affinity chromatography purification system (QIA Express, Qiagen).
[0085]
Example 3
Use of essential coding sequence products in screening for antimicrobial agents
Individual purified proteins (ie, target proteins) are combined with the sample and screened for ligands that bind to the target protein. The method used for screening is described in US Pat. No. 5,783,397 to Hughes et al. Screening is performed by Cetek Corporation, Narlborough, MA.
[0086]
Example 4
Cloning of S. aureus uridylate kinase coding sequence for expression in E. coli
The S. aureus pyrH coding sequence coding for uridylate kinase is cloned into the expression vector pQE60 for production of recombinant uridylate kinase in E. coli strain M15 containing plasmid pREP4. Cloning of the pyrH coding sequence was by PCR with two oligonucleotide primers 5'CCCGGGCCATGGCTCAAATT (SEQ ID NO: 90) and 5'GGGCCCAAGCTTAGTGATGG (SEQ ID NO: 145) using S. aureus genomic DNA as a template. . The PCR product is treated with restriction enzymes NcoI and HindIII, purified by agarose gel electrophoresis and ligated to pQE60 digested with NcoI and HindIII. The ligation mixture was transformed into M15 cells containing pREP4; transformants were selected and the nucleotide sequence of the pyrH coding sequence was confirmed by restriction enzyme analysis and DNA sequencing. The resulting plasmid for the production of S. aureus urgyrate kinase in E. coli was named pQE60-UMK. Techniques for DNA and plasmid preparation, restriction enzyme treatment, ligation, and transformation were as described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)).
[0087]
The nucleotide and amino acid sequence of recombinant uridylate kinase is shown in FIG. Six histidine residues are added to the C-terminus for purification by Ni-chelating chromatography. For use of the NcoI site and vector sequence for cloning, an alanine residue is added after the start methionine, and an arginine and serine residue is added prior to the histidine residue.
[0088]
Example 5
Production of recombinant S. aureus urdylate kinase
A production strain designated N15 (pQE60-UMK) was obtained at 30 ° C. with NS86 medium at about 1 A. ₅₅₀ Allowed to grow. NS86 medium is 2.6g K in 1 liter of water ₂ HPO _Four 10.9 g NaNH _Four HPO _Four -4H ₂ O, 2.1 g citric acid, 0.67 g (NH _Four ) ₂ SO _Four 0.25 g MgSO _Four -7H ₂ O, 10.4 g yeast extract and 5 g glycerol. NS86 medium was supplemented with 100 μg / ml ampicillin and 25 μg / ml kanamycin. A frozen ampoule with 20% glycerol added as a cryoprotectant was prepared and stored in liquid nitrogen.
[0089]
Seed cultures were prepared by inoculation of 0.1 ml thawed cells from ampules into 50 ml NS86 medium, grown overnight at 30 ° C., and used to inoculate 100 ml of MIM medium. MIM medium is A ₅₅₀ 32 g tryptone, 20 g yeast extract, 6 g Na in 1 liter of water containing up to 0.1 100 μg / ml ampicillin and 25 μg / ml kanamycin ₂ HPO _Four 3g KH ₂ PO _Four 0.5 g NaCl and 1 g NH _Four Cl. Cells are A overnight at 30 ° C. ₅₅₀ Grown to 7-8 and used in 10 liter fermentation.
[0090]
Using seed culture, A ₅₅₀ A 10 liter fermenter (New Brunswick Microgens) containing MIM medium to 0.1 was inoculated. Cells are about 1 A at 30 ° C ₅₅₀ When grown to a density of 1, isopropyl-β-D-thiogalactoside was added to 1 mM to induce expression of the recombinant protein. Cells were harvested 2 hours after induction and stored frozen. The average amount of uridylate kinase resulting from 10 liters of fermentation is estimated at 170 mg / l, which corresponds to about 20-25% of the total cellular protein.
[0091]
Example 6
Purification of recombinant S. aureus uridylate kinase
Thaw frozen cells from 10 liter fermentation and 200 ml cold lysis buffer (50 mM Tris, pH 7.8 at 22 ° C., 500 mM NaCl, 10% glycerol, 25 mM imidazole, 5 mM 2-mercaptoethanol, 0.1 mg / ml DNase ). The pellet was homogenized to obtain a uniform suspension, which was treated twice with a Rainie homogenizer to lyse the cells. Lysed cells were centrifuged at 35,000 × g for 75 minutes, and the supernatant liquid was sequentially filtered through Nalgene 0.45 micron and 0.2 micron filters to remove particulate matter prior to column chromatography.
[0092]
Column chromatography was performed at 4 ° C. using a 2.6 cm × 6.7 cm column packed with Qiagen Ni-NTA Superflow resin. The column was washed with 3 bed volumes of water and equilibrated with 3 bed volumes of equilibration buffer (50 mM Tris, pH 7.8 at 22 ° C., 500 mM NaCl, 10% glycerol, 5 mM 2-mercaptoethanol) containing 25 mM imidazole. Turned into. The filtered supernatant was applied to the column at a rate of 3 bed volumes per hour. After loading, the column was washed with equilibration buffer containing 25 mM imidazole until the absorbance at 280 nanometers (nm) decreased to 50% of baseline, followed by 6-7 bed volumes of equilibration buffer + 40 mM imidazole. It was then washed with 6-7 bed volumes of equilibration buffer + 50 mM imidazole. Eluted uridylate kinase bound in equilibration buffer + 300 mM imidazole at a rate of 2 to 3 bed volumes per hour, recovered in 4 separate fractions, pooled and diluted 3 fold to increase protein concentration Reduced and dialyzed against 50 mM Tris, 22 ° C., pH 7.8, 500 mM NaCl, 10% glycerol, 5 mM 2-mercaptoethanol. The dialyzed pool was stored frozen until further use.
[0093]
This isolation yielded approximately 700 mg of recombinant uridylate kinase protein with a purity of 95-98%. N-terminal sequencing indicates the presence of an N-terminal methionine, which is predicted to be due to the activity of the host methionine aminopeptidase.
[0094]
Example 7
Enzymatic assay for uridylate kinase activity
Uridylate kinase catalyzes the transfer of the phosphoryl group from ATP to UMP to form UDP. In cells, UDP is a substrate / precursor in several metabolic pathways including RNA and synthesis. A spectrophotometric assay was established by coupling the uridylate kinase reaction to NADH oxidation using pyruvate kinase and lactate dehydrogenase, which eventually resulted in lactate and NAD the product of the uridylate kinase reaction. ⁺ Convert to NADH oxidation was monitored by the decrease in absorbance at 340 nm. The assay was stopped with EDTA at a final concentration of 5 mM. This assay was optimized in a high-throughput format in 96-well microtiter plates to screen for agents that inhibit uridylate kinase. A second assay for pyruvate kinase and lactate dehydrogenase coupling enzyme was also developed to test the specificity of the agents detected in the primary coupled assay.
[0095]
The coupled assay is shown below.
[0096]
[Expression 1]

[0097]
Abbreviations:
ATP / ADP-adenosine 5'-triphosphate / adenosine 5'-diphosphate
UMP / UDP-uridine 5 'monophosphate / uridine 5' diphosphate
PEP-phospho (enol) pyruvate
NADH / NAD + -reduced / oxidized nicotinamide adenine dinucleotide
The uridylate kinase coupling assay contains the following reagents in a final volume of 200 μl: assay buffer (50 mM HEPES, pH 7.5, 100 mM KCl, 2 mM MgCl ₂ ), 1 mM UMP, 2 mM ATP, 0.22 mM NADH, 2 mM PEP, 3.2 units pyruvate kinase (Sigma, St. Louis, MO), 4 units lactate dehydrogenase (Sigma) and 10 ng uridylate kinase. The assay was performed at 25 ° C. and monitored at 15 second intervals in a 1 or 3 hour dynamic mode and the absorbance decreased at 340 nm.
[0098]
The complete disclosure of all patents, patent applications, publications, and nucleic acid and protein database entries cited herein, including, for example, GenBank accession numbers and EMBL accession numbers, are here as if incorporated herein. Quote and integrate. Various modifications and variations of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention, and it should be understood that the invention is not limited to the exemplary embodiments described herein. It is.
[0099]
[Chemical formula 2]

[Brief description of the drawings]
[0100]
1a-z: Nucleotide sequences of 26 S. aureus coding sequences (SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33,109,113,117,121,125,129,133,137 and 141), the predicted sequences of the peptides encoded by each coding sequence (SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, respectively) 26, 28, 30, 32, 34, 110, 114, 118, 122, 126, 130, 134, 138 or 142), and primer pairs used to prepare fragments for insertion into temperature sensitive plasmids (SEQ ID NOs: 35-68, 111-112, 115-116, 119-120, 123-124, 127-128, 131- 132,135-136,139-140 and 143-144). The two underlined sequences in each coding sequence correspond to the primers listed below the coding sequence.
Figures 2a-i: Nucleotide sequences of each of the nine S. aureus coding sequences to be cloned for expression in E. coli (SEQ ID NOs: 69, 71, 73, 75, 77, 79, 81, 83 and 85). The predicted sequence of the peptide encoded by each coding sequence (SEQ ID NO: 70, 72, 74, 76, 78, 80, 82, 84 and 86, respectively) after insertion into the appropriate expression plasmid, and by amplification Sequence of primer pair used to clone S. aureus coding sequence (SEQ ID NO: 91 to 108). The top and bottom primers of each primer pair are a forward primer and a reverse primer, respectively. The underlined ATGG in SEQ ID NOs: 69, 73, 75, 77 and 79 indicates the position of a portion of the NcoI restriction site to which the coding sequence was added by the forward primer for cloning into the expression vector pQE-60. AGATCT underlined in SEQ ID NOs: 69, 71, 73, 75, 79, 81, 83 and 85 indicates the position of the BglII restriction site added to the coding sequence by the reverse primer. GGATCT underlined in SEQ ID NO: 77 indicates the position of ligation between the digested BglII restriction site of the vector and the digested BamHI restriction site of the amplified fragment.
FIG. 3 shows the nucleotide sequence of S. aureus uridylate kinase (column number: 87) and the predicted amino acid sequence (SEQ ID NO: 88).

Claims (55)

A method for identifying an agent that binds to a polypeptide comprising the steps of:
A polypeptide and an agent are contacted to form a mixture, wherein the polypeptide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, 137 and 141. Encoded by a coding sequence comprising:
Determining the agent binding to the polypeptide. The method of claim 1, wherein the determination comprises an assay selected from the group consisting of an enzyme assay, a binding assay, and a ligand binding assay. And contacting the agent with a microorganism;
Incubating the microorganism and the agent under conditions suitable for growth of the microorganism not contacted with the agent;
Determining the growth rate of the microorganism in contact with the agent, wherein a decrease in the growth rate compared to a microorganism not in contact with the agent indicates that the agent decreases the growth rate of the microorganism;
The method of claim 1, comprising determining whether the agent reduces the growth rate of the microorganism. 4. The method of claim 3, wherein the microorganism is in vitro or in vivo. The method according to claim 3, wherein the microorganism is Staphylococcus aureus. An agent identified by the method of claim 1. A method for identifying an agent that binds to a polypeptide, comprising:
A polypeptide and an agent are contacted to form a mixture, wherein the polypeptide is a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133 and 137. Encoded by an essential coding sequence comprising a nucleotide sequence having at least about 57 percent structural similarity to;
Determining the agent binding to the polypeptide. 8. The method of claim 7, wherein the determination comprises an assay selected from the group consisting of an enzyme assay, a binding assay, and a ligand binding assay. And contacting the agent with a microorganism;
Incubating the microorganism and the agent under conditions suitable for growth of microorganisms not in contact with the agent;
Determining the growth rate of the microorganism in contact with the agent, wherein a decrease in the growth rate compared to a microorganism not in contact with the agent indicates that the agent decreases the growth rate of the microorganism;
8. The method of claim 7, comprising determining whether the agent reduces the growth rate of the microorganism. 10. The method of claim 9, wherein the microorganism is in vitro or in vivo. The method according to claim 9, wherein the microorganism is S. aureus. An agent identified by the method of claim 9. A method for identifying an agent that binds to a polypeptide comprising the steps of:
Contacting the polypeptide and the agent to form a mixture, wherein the polypeptide is encoded by a critical coding sequence comprising at least about 57 percent structural similarity to the nucleotide sequence comprising SEQ ID NO: 141;
Determining the agent binding to the polypeptide. 14. The method of claim 13, wherein the determination comprises an assay selected from the group consisting of an enzyme assay, a binding assay, and a ligand binding assay. And contacting the agent with a microorganism;
Incubating the microorganism and the agent under conditions suitable for growth of microorganisms not in contact with the agent;
Determining the growth rate of a microorganism in contact with the agent, wherein a decrease in growth rate compared to a microorganism not in contact with the agent indicates that the agent decreases the growth rate of the microorganism;
14. The method of claim 13, comprising determining whether the agent comprising reducing the growth rate of the microorganism. 14. The method of claim 13, wherein the microorganism is in vitro or in vivo. The method according to claim 13, wherein the microorganism is S. aureus. An agent identified by the method of claim 13. A method for identifying an agent that reduces the growth rate of a microorganism, comprising:
Contacting the microorganism with an agent, wherein the agent is encoded by a coding sequence comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, 137 and 141 Binds to a polypeptide;
Incubating the microorganism and the agent under conditions suitable for the growth of microorganisms not in contact with the agent; then determining the growth rate of the microorganism in contact with the agent, wherein the microorganism not in contact with the agent A decrease in growth rate compared to indicates that the agent decreases the growth rate of the microorganism;
The method comprising: 20. The method of claim 19, wherein the microorganism is in vitro or in vivo. The method according to claim 19, wherein the microorganism is S. aureus. 20. An agent identified by the method of claim 19. A method for identifying an agent that reduces the growth rate of a microorganism, comprising:
Contacting the microorganism with an agent, wherein the agent is at least about 57 percent relative to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133 and 137. Encoded by an essential coding sequence comprising a nucleotide sequence with structural similarity;
Incubating the microorganism and the agent under conditions suitable for growth of microorganisms not in contact with the agent;
Determining the growth rate of a microorganism in contact with the agent, wherein a decrease in growth compared to a microorganism not in contact with the agent indicates that the agent decreases the growth rate of the microorganism;
The method comprising: 24. The method of claim 23, wherein the microorganism is in vitro or in vivo. The method according to claim 23, wherein the microorganism is S. aureus. 24. An agent identified by the method of claim 23. A method for identifying an agent that reduces the growth rate of a microorganism, comprising:
Contacting the microorganism with an agent, wherein the agent is encoded by a critical coding sequence comprising a nucleotide sequence having at least about 57 percent structural similarity to the nucleotide sequence comprising SEQ ID NO: 141;
Incubating the microorganism and the agent under conditions suitable for growth of microorganisms not in contact with the agent;
Determining the growth rate of the microorganism in contact with the agent, wherein a decrease in growth rate compared to a microorganism not in contact with the agent indicates that the agent decreases the growth rate of the microorganism;
The method comprising: 28. The method of claim 27, wherein the microorganism is in vitro or in vivo. 28. The method of claim 27, wherein the microorganism is S. aureus. 28. An agent identified by the method of claim 27. Contacting a microorganism with an agent that binds to a polypeptide encoded by a coding sequence comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, 137 and 141. A method for reducing the growth rate of microorganisms characterized by 32. The method of claim 31, wherein the microorganism is in vitro or in vivo. The method according to claim 31, wherein the microorganism is S. aureus. By an essential coding sequence comprising a nucleotide sequence having at least about 57 percent structural similarity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133 and 137 A method of reducing the growth rate of a microorganism, comprising contacting the microorganism with an agent that binds to the encoded polypeptide. 35. The method of claim 34, wherein the microorganism is in vitro or in vivo. 35. The method of claim 34, wherein the microorganism is S. aureus. Contacting the microorganism with an agent that binds to a polypeptide encoded by a critical coding sequence comprising a nucleotide sequence having at least about 57 percent structural similarity to the nucleotide sequence comprising SEQ ID NO: 141. A method of reducing the growth rate of microorganisms. 38. The method of claim 37, wherein the microorganism is in vitro or in vivo. 40. The method of claim 38, wherein the microorganism is S. aureus. The coding sequence in S. aureus is modified to contain a mutation and the non-mutated coding sequence is selected from the group consisting of SEQ ID NOs: 7, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, 137 and 141 A nucleotide sequence to be
A method for producing S. aureus with reduced virulence, characterized in that it is determined whether S. aureus comprising said mutation has a reduced virulence compared to S. aureus not comprising said mutation. 41. S. aureus according to claim 40. 41. A vaccine composition comprising the S. aureus organism of claim 40. The essential coding sequence in S. aureus is modified to contain a mutation, and the non-mutated coding sequence is from the group consisting of SEQ ID NOs: 7, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133 and 137 A nucleotide sequence having at least about 57 percent structural similarity to the selected nucleotide sequence;
A method for producing S. aureus with reduced virulence, characterized in that it is determined whether S. aureus comprising said mutation has a reduced virulence compared to S. aureus not comprising said mutation. 44. Aureus according to claim 43. 44. A vaccine composition comprising the S. aureus organism of claim 43. The critical coding sequence in S. aureus is modified to contain a mutation, and the non-mutated coding sequence has at least about 57 percent structural similarity to the nucleotide sequence comprising SEQ ID NO: 141 Comprising a sequence;
A method for producing S. aureus with reduced virulence, characterized in that it is determined whether S. aureus comprising said mutation has a reduced virulence compared to S. aureus not comprising said mutation. S. aureus according to claim 46. 49. A vaccine composition comprising the S. aureus organism of claim 46. An isolated polypeptide comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, 137 and 141. An isolated polypeptide consisting essentially of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, 137 and 141, wherein the polynucleotide is desired Further comprising zero to about 5,000 nucleotides upstream and / or downstream of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 7, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, 137 and 141. Polynucleotide. An isolated poly comprising a nucleotide sequence having at least about 57 percent structural similarity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7, 21, 23, 25, 27, 29, 31, 109, 113, 117, 121, 125, 129, 133 and 137 A polynucleotide, wherein the isolated polynucleotide comprises an essential coding sequence. SEQ ID NO: 7, 21, 23, 25, 27, 29, 31, 109, 113, 117, 121, 125, 129, 133 and 137 substantially isolated from a nucleotide sequence having at least about 57 percent structural similarity to a nucleotide sequence selected from the group consisting of The isolated polynucleotide, wherein the isolated polynucleotide comprises an essential coding sequence. An isolated polynucleotide comprising a nucleotide sequence having at least about 57 percent structural similarity to a nucleotide sequence comprising SEQ ID NO: 141, wherein the isolated polynucleotide comprises a critical coding sequence The polynucleotide. An isolated polynucleotide consisting essentially of a nucleotide sequence having at least about 57 percent structural similarity to a nucleotide sequence comprising SEQ ID NO: 141, wherein the isolated polynucleotide is critical coding The polynucleotide comprising a sequence. SEQ ID NO: 2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,110,114,118,122,126,130,134,138 and 142 Released polypeptide.

Images